Thermococcus kodakarensis (KOD) DNA polymerase variants with improved properties and methods of use thereof

Abstract

This disclosure describes Thermococcus kodakarensis (KOD) polymerase variants and methods of use thereof in amplifying polynucleotides. Disclosed KOD polymerase variants include KOD polymerase variants having altered salt tolerance and / or altered uracil tolerance.

Description

[0001] THERMOCOCCUS KODAKARENSIS (KOD) DNA POLYMERASE VARIANTS WITH IMPROVED PROPERTIES AND METHODS OF USE THEREOF

[0002] RELATED APPLICATIONS

[0003] This application claims benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 691,098, filed September 5, 2024, entitled “Thermococcus Kodakarensis (KOD) DNA Polymerase Variants with Improved Properties and Methods of Use Thereof’, the entire contents of which is incorporated herein by reference.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (W109470016WO00-SEQ-ARM.xml; Size: 213,240 bytes; and Date of Creation: August 29, 2025) are herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] DNA polymerases play a central role in modern molecular biology. DNA polymerases make new copies of DNA strands using an already existing DNA strand as a template. Additionally, some DNA polymerases have proof reading / repair domains that can repair errors that occur when new copies of the DNA strand are made. DNA polymerases are critical in many applications including the polymerase chain reaction (PCR), DNA sequencing, DNA cloning, synthetic biology, and molecular diagnostics. In a natural system, DNA polymerases operate in cellular physiological conditions and amplify naturally occurring DNA (typically as part of cell replication). However, in molecular biology applications, DNA polymerases may need to operate in conditions that are not physiological or be able to amplify a polynucleotide having nucleotides that do not naturally occur in DNA.

[0008] Thermococcus kodakarensis (KOD) polymerase is a thermostable DNA polymerase that is used in some molecular biology applications, like PCR which requires drastically changing temperatures (e.g., frequent changes between 60 °C to 95 °C).

[0009] SUMMARY

[0010] This disclosure provides numerous KOD polymerase variants that have altered properties (e.g., increased activity, increased activity in high salt conditions, increased activity in low salt conditions, increased uracil template tolerance (e.g., amplifying a DNA template comprising uracil), increased uracil template stringency, and altered magnesium ion tolerance) relative to wildtype KOD DNA polymerase. These KOD polymerase variants were identified by screening the function of thousands of different KOD polymerase variants in a variety of different salts

[0011] #14338400v1 and salt concentrations, and in amplifying DNA polynucleotides comprising uracil. The screens included measuring the function of thousands of mutations at more than 700 KOD polymerase amino acid positions. Results from the screen included 15 positions in KOD polymerase, that when mutated to certain amino acids, increased KOD polymerase activity in one or more of the salt conditions / concentrations (salt tolerant mutations): E325, S347, F356, L396, 1488, F587, V589, T605, G634, R641, E664, A675, G677, A747, and F748. Results also included four positions in KOD polymerase, that when mutated to certain amino acids, increased KOD polymerase activity when amplifying DNA polynucleotides comprising uracil (uracil tolerance mutations): 116, P36, P90 and V93.

[0012] In some aspects, this disclosure provides a Thermococcus kodakarensis (KOD) polymerase variant, comprising: (i) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to E325 of SEQ ID NO: 1; (ii) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, leucine, lysine, phenylalanine, proline, threonine, tryptophan, tyrosine, or valine at a position corresponding to S347 of SEQ ID NO: 1; (iii) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to F356 of SEQ ID NO: 1; (iv) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to L396 of SEQ ID NO: 1; (v) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to 1488 of SEQ ID NO: 1; (vi) an arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, methionine, proline, serine, tryptophan, or tyrosine at a position corresponding to F587 of SEQ ID NO: 1; (vii) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, or tyrosine at a position corresponding to V589 of SEQ ID NO: 1; (viii) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, or valine at a position corresponding to T605 of SEQ ID NO: 1; (ix) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or

[0013] #14338400v1 valine at a position corresponding to G634 of SEQ ID NO: 1; (x) an alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to R641 of SEQ ID NO: 1; (xi) an alanine, arginine, asparagine, aspartic acid, glycine, histidine, isoleucine, leucine, methionine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to E664 of SEQ ID NO: 1; (xii) an arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to A675 of SEQ ID NO: 1; (xiii) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to G677 of SEQ ID NO: 1; (xiv) an arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to A747 of SEQ ID NO: 1; (xv) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to F748 of SEQ ID NO: 1.

[0014] In some embodiments, the KOD polymerase variant comprising: (i) a methionine, threonine, lysine, serine, arginine, leucine, proline, histidine, glutamine, valine, alanine, glycine, phenylalanine, cysteine, or tyrosine at a position corresponding to E325 of SEQ ID NO: 1; (ii) a threonine, asparagine, arginine, lysine, glutamine, or valine at a position corresponding to S347 of SEQ ID NO: 1; (iii) an isoleucine, methionine, asparagine, lysine, serine, arginine, leucine, valine, alanine, aspartic acid, glycine, or cysteine at a position corresponding to F356 of SEQ ID NO: 1; (iv) a threonine, lysine, serine, arginine, proline, histidine, glutamine, alanine, aspartic acid, glutamic acid, glycine, tyrosine, or cysteine at a position corresponding to L396 of SEQ ID NO: 1; (v) an arginine, asparagine, lysine, glutamine, alanine, aspartic acid, or glycine at a position corresponding to 1488 of SEQ ID NO: 1; (vi) a methionine, asparagine, lysine, histidine, glutamine, tyrosine, cysteine, or glycine at a position corresponding to F587 of SEQ ID NO: 1; (vii) an asparagine, arginine, alanine, glycine, phenylalanine, tyrosine, or tryptophan at a position corresponding to V589 of SEQ ID NO: 1; (viii) an isoleucine, proline, histidine, valine, alanine, phenylalanine, tyrosine, cysteine, or tryptophan at a position corresponding to T605 of SEQ ID NO: 1; (ix) an asparagine, lysine, arginine, leucine, proline, glutamine, aspartic acid, glutamic acid, phenylalanine, tyrosine, cysteine, tryptophan, isoleucine, threonine, serine, histidine, valine, or alanine at a position corresponding to G634 OF SEQ ID NO: 1; (x) a

[0015] #14338400v1 proline, glutamic acid, or alanine at a position corresponding to R641 of SEQ ID NO: 1; (xi) a methionine, threonine, asparagine, serine, arginine, valine, or isoleucine at a position corresponding to E664 of SEQ ID NO: 1; (xii) an isoleucine, threonine, lysine, arginine, histidine, valine, or tryptophan at a position corresponding to A675 of SEQ ID NO: 1; (xiii) a methionine, threonine, serine, arginine, leucine, proline, histidine, glutamine, phenylalanine, lysine, or alanine at a position corresponding to G677 of SEQ ID NO: 1; (xiv) a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at a position corresponding to A747 of SEQ ID NO: 1; and / or (xv) a lysine, arginine, histidine, aspartic acid, tyrosine, tryptophan, or serine at a position corresponding to F748 of SEQ ID NO: 1.

[0016] In some embodiments, the KOD polymerase variant comprises a methionine, threonine, lysine, serine, arginine, leucine, proline, histidine, glutamine, valine, alanine, glycine, phenylalanine, cysteine, or tyrosine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a valine at a position corresponding to E325 of SEQ ID NO: 1.

[0017] In some embodiments, the KOD polymerase variant comprises a threonine, asparagine, arginine, lysine, glutamine, or valine at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an arginine at a position corresponding to S347 of SEQ ID NO: 1.

[0018] In some embodiments, the KOD polymerase variant comprises an isoleucine, methionine, asparagine, lysine, serine, arginine, leucine, valine, alanine, aspartic acid, glutamine, or cysteine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an asparagine at a position corresponding to F356 of SEQ ID NO: 1.

[0019] In some embodiments, the KOD polymerase variant comprises a threonine, lysine, serine, arginine, proline, histidine, glutamine, alanine, aspartic acid, glutamic acid, glycine, tyrosine, or cysteine at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a glutamic acid at a position corresponding to L396 of SEQ ID NO: 1.

[0020] In some embodiments, the KOD polymerase variant comprises an arginine, asparagine, lysine, glutamine, alanine, aspartic acid, or glycine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an arginine at a position corresponding to 1488 of SEQ ID NO: 1.

[0021] #14338400v1 In some embodiments, the KOD polymerase variant comprises a methionine, asparagine, lysine, histidine, glutamine, tyrosine, cysteine, or glycine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a methionine at a position corresponding to F587 of SEQ ID NO: 1.

[0022] In some embodiments, the KOD polymerase variant comprises an asparagine, arginine, alanine, glycine, phenylalanine, tyrosine, or tryptophan at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a tryptophan at a position corresponding to V589 of SEQ ID NO: 1.

[0023] In some embodiments, the KOD polymerase variant comprises an isoleucine, proline, histidine, valine, alanine, phenylalanine, tyrosine, cysteine, or tryptophan at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a tyrosine at a position corresponding to T605 of SEQ ID NO: 1.

[0024] In some embodiments, the KOD polymerase variant comprises an asparagine, lysine, arginine, leucine, proline, glutamine, aspartic acid, glutamic acid, phenylalanine, tyrosine, cysteine, tryptophan, isoleucine, threonine, serine, histidine, valine, or alanine at a position corresponding to G634 OF SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an aspartic acid at a position corresponding to G634 OF SEQ ID NO: 1.

[0025] In some embodiments, the KOD polymerase variant comprises a proline, glutamic acid, or alanine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a proline at a position corresponding to R641 of SEQ ID NO: 1.

[0026] In some embodiments, the KOD polymerase variant comprises a methionine, threonine, asparagine, serine, arginine, valine, or isoleucine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a lysine at a position corresponding to E664 of SEQ ID NO: 1.

[0027] In some embodiments, the KOD polymerase variant comprises an isoleucine, threonine, lysine, arginine, histidine, valine, or tryptophan at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an arginine at a position corresponding to A675 of SEQ ID NO: 1.

[0028] In some embodiments, the KOD polymerase variant comprises a methionine, threonine, serine, arginine, leucine, phenylalanine, histidine, glutamine, proline, lysine, or alanine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a proline at a position corresponding to G677 of SEQ ID NO: 1.

[0029] #14338400v1 In some embodiments, the KOD polymerase variant comprises a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1.

[0030] In some embodiments, the KOD polymerase variant comprises a lysine, arginine, histidine, aspartic acid, tyrosine, tryptophan, or serine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a tyrosine at a position corresponding to F748 of SEQ ID NO: 1.

[0031] In some embodiments, the KOD polymerase variant comprises one or more of: an isoleucine, leucine or valine at a position corresponding to F587 of SEQ ID NO: 1; a histidine or glutamine at a position corresponding to V589 of SEQ ID NO: 1; a lysine, glutamine, or cysteine at a position corresponding to E664 of SEQ ID NO: 1; or a methionine at a position corresponding to A675 of SEQ ID NO: 1.

[0032] In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 2-6. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 7-9. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NOs: 10. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 11-23. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 24. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 25-34. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 35- 43. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 44-52. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 53-70. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 71-72. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 73-80. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 81-88. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 89-97. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 98-105. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 106-111.

[0033] #14338400v1 In some embodiments, the KOD polymerase variant comprises: (i) a lysine at a position corresponding to E664 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1; (ii) a tyrosine at a position corresponding to T605 and a lysine at a position corresponding to E664 of SEQ ID NO: 1; (iii) a methionine at a position corresponding to F587 and a lysine at a position corresponding to E664 of SEQ ID NO: 1; (iv) an arginine at a position corresponding to S347 and a lysine at a position corresponding to E664 of SEQ ID NO: 1; (v) a proline at a position corresponding to G677 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1; (vi) an arginine at a position corresponding to A675 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1; (vii) an arginine at a position corresponding to S347 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1; or (viii) an arginine at a position corresponding to S347, a lysine at a position corresponding to E664, and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a lysine at a position corresponding to E664 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1.

[0034] In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 112. In some embodiments, the KOD polymerase variant comprises a tyrosine at a position corresponding to T605 and a lysine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 113.

[0035] In some embodiments, the KOD polymerase variant comprises a methionine at a position corresponding to F587 and a lysine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 114.

[0036] In some embodiments, the KOD polymerase variant comprises an arginine at a position corresponding to S347 and a lysine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 115.

[0037] In some embodiments, the KOD polymerase variant comprises a proline at a position corresponding to G677 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 116.

[0038] In some embodiments, the KOD polymerase variant comprises an arginine at a position corresponding to A675 and an aspartic acid at a position corresponding to A747 of SEQ ID NO:

[0039] #14338400v1 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 117.

[0040] In some embodiments, the KOD polymerase variant comprises an arginine at a position corresponding to S347 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 118.

[0041] In some embodiments, the KOD polymerase variant comprises an arginine at a position corresponding to S347, a lysine at a position corresponding to E664, and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 119.

[0042] In some embodiments, the KOD polymerase variant comprises a mutation at a position corresponding to one or more of 116, P36, P90, or V93 of SEQ ID NO: 1.

[0043] In some embodiments, the KOD polymerase variant comprises: (i) an alanine, cysteine, glutamic acid, phenylalanine, histidine, lysine, asparagine, proline, serine, valine, tryptophan, or tyrosine at a position corresponding to 116 of SEQ ID NO: 1; (ii) an aspartic acid, glutamic acid, phenylalanine, isoleucine, leucine, methionine, asparagine, glutamine, valine, tryptophan, tyrosine at a position corresponding to P36 of SEQ ID NO: 1; (iii) a cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, arginine, tryptophan, or tyrosine at a position corresponding to P90 of SEQ ID NO: 1; and / or (iv) alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, tryptophan, or tyrosine at a position corresponding to V93 of SEQ ID NO: 1.

[0044] In some embodiments, a KOD polymerase variant comprises: (i) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to 116 of SEQ ID NO: 1; (ii) an alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to P36 of SEQ ID NO: 1; (iii) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to P90 of SEQ ID NO: 1; and / or (iv) an alanine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, or tyrosine at a position corresponding to V93 of SEQ ID NO: 1.

[0045] #14338400v1 In some embodiments, the KOD polymerase variant comprises: (i) an alanine, cysteine, glutamic acid, phenylalanine, histidine, lysine, asparagine, proline, serine, valine, tryptophan, or tyrosine at a position corresponding to 116 of SEQ ID NO: 1; (ii) an aspartic acid, glutamic acid, phenylalanine, isoleucine, leucine, methionine, asparagine, glutamine, valine, tryptophan, tyrosine at a position corresponding to P36 of SEQ ID NO: 1; (iii) a cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, arginine, tryptophan, or tyrosine at a position corresponding to P90 of SEQ ID NO: 1; and / or (iv) alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, leucine, methionine, asparagine, proline, serine, threonine, tryptophan, or tyrosine at a position corresponding to V93 of SEQ ID NO: 1.

[0046] In some embodiments, the KOD polymerase variant comprises an alanine, cysteine, glutamic acid, phenylalanine, histidine, lysine, asparagine, proline, serine, valine, tryptophan, or tyrosine at a position corresponding to 116 of SEQ ID NO: 1.

[0047] In some embodiments, the KOD polymerase variant comprises a tryptophan at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an aspartic acid, glutamic acid, phenylalanine, isoleucine, leucine, methionine, asparagine, glutamine, valine, tryptophan, tyrosine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, arginine, tryptophan, or tyrosine at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a tryptophan at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, leucine, methionine, asparagine, proline, serine, threonine, tryptophan, or tyrosine at a position corresponding to V93 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises: (i) a histidine, lysine or arginine at a position corresponding to P36 of SEQ ID NO: 1; and (ii) a histidine, lysine, glutamine, or arginine at a position corresponding to V93 of SEQ ID NO: 1.

[0048] In some embodiments, a KOD polymerase variant, comprises: (i) an arginine at a position corresponding to S347 of SEQ ID NO: 1; or (ii) an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises the arginine at the position corresponding to S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises the aspartic acid at the position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a lysine at the position corresponding to E664 of SEQ ID NO: 1. In some

[0049] #14338400v1 embodiments, the KOD polymerase variant comprises the aspartic acid at the position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a lysine at position corresponding to E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises: (i) the arginine at the position corresponding to S347 of SEQ ID NO: 1; (ii) the aspartic acid at the position corresponding to A747 of SEQ ID NO: 1; and (ii) a lysine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises: (a) a glutamine at a position corresponding to V93 of SEQ ID NO: 1; (b) a tryptophan at a position corresponding to P90 of SEQ ID NO: 1; or (c) a tryptophan at a position corresponding to 116 of SEQ ID NO: 1.

[0050] In some embodiments, the KOD polymerase variant comprises the glutamine at the position corresponding to V93 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises the tryptophan at the position corresponding to P90 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises the tryptophan at the position corresponding to 116 of SEQ ID NO: 1.

[0051] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 9, 102, 112, or 115-119. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 9, 102, 112, or 115-119. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 120- 122. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 120-122. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 120. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 121. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 122.

[0052] In some embodiments, the KOD polymerase variant comprises (i) the tryptophan at the position corresponding to P90 of SEQ ID NO: 1; (ii) the tryptophan at the position corresponding to 116 of SEQ ID NO: 1; and (iii) a glutamine at a position corresponding to V93 of SEQ ID NO: 1.

[0053] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 120-125. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 120- 125. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2-125. In some embodiments, this

[0054] #14338400v1 disclosure provides a polynucleotide variant comprising a nucleic acid encoding a KOD polymerase variant disclosed herein.

[0055] In some embodiments, this disclosure provides a method for nucleic acid synthesis, the method comprising contacting a nucleic acid template with primers, deoxynucleotide triphosphates (dNTPs), and a KOD polymerase variant described herein. In some embodiments, the nucleic acid synthesis is performed in more than 150 mM of potassium ion. In some embodiments, the nucleic acid synthesis is performed in at least 300 mM of potassium ion. In some embodiments, the nucleic acid synthesis is performed in less than 1.5 mM of magnesium ion.

[0056] BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIGs. 1A-1B show identification of single- site Thermococcus kodakarensis (KOD) polymerase variants that alter uracil tolerance. FIG. 1A shows the relative activity of KOD polymerase variants representing all possible amino acid substitutions across residues 116, P36, P90, and V93, shown as a fold-change compared to wild-type KOD polymerase. * indicates p value < 0.05; V indicates substitutions that were further characterized. FIG. IB shows cycle threshold (Cq) values for wild-type KOD polymerase and KOD polymerase variants comprising single-site mutations V93Q, P90W, P36W, or I16W at 0.5 ng / pL, 2 ng / pL, 5 ng / pL, or 7.5 ng / pL of enzyme, dUTP / dTTP ratios of 0%, 50%, or 100% in the nucleotide pool, and in 100 mM KC1. For FIG. IB, the legend indicating dUTP / dTTP % applies to the bars of each group from left to right.

[0058] FIGs. 2A-2C show identification of single-site KOD polymerase variants that exhibit altered salt tolerance. FIG. 2A shows the relative activity of KOD polymerase variants representing all possible amino acid substitutions across residues E325, S347, F356, L396, 1488, F587, V589, T605, G634, R641, E664, A675, G677, A747, and F748, shown as a fold-change in relative activity compared to wild-type KOD polymerase in 300 mM KC1. * indicates p value < 0.05; V indicates substitutions that were further characterized. FIG. 2B shows the relative activity of KOD polymerase variants representing all possible amino acid substitutions across residues E325, S347, F356, L396, 1488, F587, V589, T605, G634, R641, E664, A675, G677, A747, and F748, shown as a fold-change compared to wild-type KOD polymerase in 1 mM Mg. * indicates p value < 0.05; V indicates substitutions that were further characterized. FIG. 2C shows Cqvalues for wild-type KOD polymerase and KOD polymerase variants comprising single-site mutations V589W, E325V, S347R, F356N, L396E, I488R, F587M, F748Y, R641P,

[0059] #14338400v1 G634D, T605Y, A675R, A747D, G677P, or E664K at 3.8 ng / pE of enzyme in the presence of 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 250 mM, or 300 mM KC1. For FIG. 2C, the legend indicating the enzyme applies to the bars of each group from left to right.

[0060] FIGs. 3A-3E show further improved salt tolerance of KOD polymerase variants comprising a double mutation. FIG. 3A shows Cqvalues for KOD polymerase variants comprising single-site mutations A747D or E664K and KOD polymerase variants comprising a A747D / E664K double mutation at 0.5 ng / pL of enzyme in the presence of 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, or 250 mM KC1. FIG. 3B shows Cqvalues for wild- type KOD polymerase, KOD polymerase variants comprising single- site mutations S347R or E664K, and KOD polymerase variants comprising a S347R / E664K double mutation at 0.9 ng / pL of enzyme in the presence of 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 250 mM, or 300 mM KC1. FIG. 3C shows Cqvalues for wild-type KOD polymerase, KOD polymerase variants comprising single-site mutations S347R or A747D, and KOD polymerase variants comprising a S347R / A747D double mutation at 0.9 ng / pL of enzyme in the presence of 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, or 175 mM KC1. FIG. 3D shows Cqvalues for wild-type KOD polymerase, KOD polymerase variants comprising single-site mutations F587M or E664K, and KOD polymerase variants comprising a F587M / E664K double mutation at 0.9 ng / pL of enzyme in the presence of 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 250 mM, or 300 mM KC1. FIG. 3E shows Cqvalues for wild- type KOD polymerase, KOD polymerase variants comprising single- site mutations G677P, A675R, or A747D, and KOD polymerase variants comprising a A675R / A747D or G677P / A747D double mutation at 0.9 ng / pL of enzyme in the presence of 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, or 200 mM KC1. For FIGs. 3A- 3E, the legend indicating the enzyme applies to the bars of each group from left to right.

[0061] FIGs. 4A-4B show the further improved salt tolerance of KOD polymerase variants comprising a triple mutation. FIG. 4A shows Cqvalues for wild-type KOD polymerase, KOD polymerase variants comprising single-site mutations S347R, A747D, or E664K, and KOD polymerase variants comprising a S347R / A747D / E664K triple mutation at 0.9 ng / pE of enzyme in the presence of 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 250 mM, 300 mM, or 400 mM KC1. FIG. 4B shows Cqvalues for wild-type KOD polymerase, KOD polymerase variants comprising single-site mutations S347R, A747D, or E664K, KOD polymerase variants comprising a E664K / A747D, S347R / A747D, or S347R / E664K double

[0062] #14338400v1 mutation, and KOD polymerase variants comprising a S347R / A747D / E664K triple mutation at 0.9 ng / pL of enzyme in the presence of 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 250 mM, 300 mM, or 400 mM KC1. For FIGs. 4A-4B, the legend indicating the enzyme applies to the bars of each group from left to right.

[0063] FIGs. 5A-5B show altered performance of KOD polymerase variants comprising a single uracil tolerance-inducing mutation and one or more salt tolerance-inducing mutations. FIG. 5A shows Cqvalues for a KOD polymerase variant comprising a single uracil toleranceinducing mutation (P90W) with or without a salt tolerance-inducing mutation (S347R, E664K, A747D, A675R, or F587M) at 0.5 ng / pE, 2 ng / pL, 5 ng / pL, or 7.5 ng / pL of enzyme and dUTP / dTTP ratios of 0%, 50%, or 100% in the presence of 100 mM or 250 mM KC1. Uracil was introduced into amplified polynucleotides via PCR primers comprising dTTP to dUTP substitutions placed towards the 3’ prime end of the molecule, such that efficient self-replication is only performed by polymerase variants that can efficiently read through uracil containing templates, as the extension products of modified primers serve as templates in subsequent cycles of PCR. FIG. 5B shows Cqvalues for a KOD polymerase variant comprising a single uracil tolerance-inducing mutation (V93Q, P90W, or I16W), a KOD polymerase variant comprising a single uracil tolerance-inducing mutation and two salt tolerance-inducing mutations (V93Q / E664K. / A747D), and KOD polymerase variants comprising a single uracil toleranceinducing mutation and three salt tolerance-inducing mutations (V93Q / S347R / E664K / A747D, P90W / S347R / E664K / A747D, or I16W / S347R / E664K / A747D) at 0.5 ng / pL, 2 ng / pL, 5 ng / pL, or 7.5 ng / pL of enzyme and dUTP / dTTP ratios of 0%, 50%, or 100% in the presence of 100 mM or 250 mM KC1. For FIGs. 5A-5B, the legend indicating the enzyme applies to the bars of each group from left to right.

[0064] FIG. 6 shows increased yield of bisulfite-treated DNA libraries by quad-site KOD polymerase mutants (V93Q / S347R / E664K / A747D, P90W / S347R / E664K / A747D, and I16W / S347R / E664K / A747D) compared to a KOD polymerase comprising a single uracil tolerance-inducing mutation (V93Q).

[0065] DETAIEED DESCRIPTION

[0066] In some aspects, this disclosure provides a KOD polymerase variant. In some embodiments, the KOD polymerase variant comprises altered salt tolerance (e.g., altered tolerance to high potassium chloride concentrations compared to wildtype and altered tolerance to low magnesium ion concentrations compared to wildtype. In some embodiments, a KOD polymerase variant comprises altered uracil tolerance (e.g., compared to wildtype KOD

[0067] #14338400v1 polymerase). For example, some KOD polymerase variants described herein amplify DNA comprising uracil with greater activity than wildtype KOD polymerase. In another example, some KOD polymerase variants described herein can introduce uracil nucleotides when amplifying a template polynucleotide. In some embodiments, a KOD polymerase variant comprises increased salt tolerance and increased uracil tolerance (e.g., compared to wildtype KOD polymerase).

[0068] A “Thermococcus kodakarensis (KOD) polymerase” refers to a family B DNA polymerase of Thermococcus kodakarensis (e.g., a wildtype family B KOD polymerase). In some embodiments, a KOD polymerase comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99% identity) to SEQ ID NO: 1. In some embodiments, a KOD polymerase comprises an amino acid sequence of SEQ ID NO: 1.

[0069] A “Thennococcus kodakarensis (KOD) polymerase variant” refers to a KOD polymerase that comprises at least one amino acid mutation relative to a KOD polymerase (e.g., relative to a wildtype KOD polymerase (SEQ ID NO: 1)). In some embodiments, a KOD polymerase variant comprises a substitution mutation relative to a KOD polymerase. In some embodiments, a KOD polymerase variant comprises one or more substitution mutations relative to a KOD polymerase. In some embodiments, a KOD polymerase variant comprises an insertion and / or deletion mutation relative to a KOD polymerase. In some embodiments, a KOD polymerase variant comprises a substitution mutation that increases salt tolerance of the KOD polymerase variant relative to a KOD polymerase (e.g., increased activity of the KOD polymerase variant in the presence of salt and / or salt concentration relative to a KOD polymerase). In some embodiments, a KOD polymerase variant comprises a substitution mutation that increases uracil tolerance of the KOD polymerase variant relative to a KOD polymerase.

[0070] In some embodiments, a KOD polymerase variant further comprising a protein tag. In some embodiments, the protein tag is a purification tag (e.g., a His tag (HHHHHH) (SEQ ID NO: 134), a Myc tag, or a Flag tag). In some embodiments, a KOD polymerase variant comprising a linker (e.g., KL) between the KOD polymerase variant and the protein tag. In some embodiments, the protein tag is a secretion tag. In some embodiments, the disclosure provide a fusion protein comprising a KOD polymerase variant and a Sso7d protein. In some embodiments, the fusion protein comprising from N-terminal to C-terminal the KOD polymerase variant and the Sso7d protein. Sso7d is a nonspecific DNA-binding protein from the crenarchaeon Sulfolobus solfataricus or a variant thereof (e.g. a protein comprising at least 95% identity to wildtype Sso7d from Sulfolobus solfataricus or a homolog of Sso7d). In some embodiments, Sso7d comprises an amino acid sequence of SEQ ID NO: 126 (

[0071] #14338400v1 (M)ATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELL QMLEKQKK), or an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity to SEQ ID NO: 126. In some embodiments, the fusion protein comprises from N-terminal to C-terminal the Sso7d protein and the KOD polymerase variant. In some embodiments, the fusion protein comprises a peptide linker between the Sso7d protein and the KOD polymerase variant. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 127 (KLGTGGGG). In some embodiments, the fusion protein comprising a KOD polymerase variant (e.g., any one of SEQ ID NOs: 2-125), a linker (e.g., SEQ ID NO: 127 and a Sso7d protein (e.g., SEQ ID NO: 126). In some embodiments, the fusion protein comprising a Sso7d protein (e.g., SEQ ID NO: 126), a linker (e.g., SEQ ID NO: 127), and KOD polymerase variant (e.g., any one of SEQ ID NOs: 2-125).

[0072] A KOD polymerase variant having a “position corresponding to” a reference amino acid sequence (e.g., a reference KOD polymerase amino acid sequence like SEQ ID NO: 1) refers to a particular amino acid in the KOD polymerase variant whose location in the amino acid sequence of the KOD polymerase variant is identified relative to the reference sequence. In other words, the reference sequence can be used as a standard for mapping mutations between different KOD polymerase variants (e.g., variants comprising insertions or deletions that shift the location of a mutation relative to the reference sequence). In some embodiments, determining a position of a KOD polymerase variant corresponding to a reference sequence comprises aligning the reference sequence and the KOD polymerase variant sequence (e.g., using Clustal Omega as described in Madeira F et al., Nucleic Acids Res. 2024 Jul;52(Wl) W521-W525. PMID: 38597606). In some embodiments, this is determined using a structural alignment (e.g., as described in Sebastian Bittrich et al., Bioinformatics, Volume 40, Issue 6, June 2024, doi.org / 10.1093 / bioinformatics / btae370). Directly below are four examples all relating to a KOD polymerase variant comprising a methionine at a position corresponding to Y38 of a reference sequence (Ref). All the below KOD polymerase variants in these four examples would fall within the scope of a KOD polymerase variant comprising a methionine at a position corresponding to Y38 of a reference sequence (Ref).

[0073] In a first example, the reference sequence (Ref) and the target sequence (Tar) align at every amino acid between positions 4 and 37 of the reference sequence. Position 38 of the target sequence corresponds to position 38 of the reference sequence. Position 38 of the target sequence is methionine. Position 38 of the reference sequence is tyrosine. (Top SEQ ID NO: 128, bottom SEQ ID NO: 129).

[0074] #14338400v1 Re f 4 DTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPY 38

[0075] Tar 4 DTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPM 38

[0076] In a second example, the target sequence has a three amino acid deletion and a tyrosine to methionine substitution relative to the reference sequence. Because of the deletion, position 35 of the target sequence corresponds to position 38 of the reference sequence. Position 35 of the target sequence is methionine. Position 38 of the reference sequence is tyrosine. (Top SEQ ID NO: 128, bottom SEQ ID NO: 130).

[0077] Re f 4-DTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPY 38

[0078] Tar 4-DT - TEDGKPVIRIFKKENGEFKIEYDRTFEPM 35

[0079] In a third example, the target sequence has a four amino acid insertion and a tyrosine to methionine substitution relative to the reference sequence. Because of the insertion, position 42 of the target sequence corresponds to position 38 of the reference sequence. Position 42 of the target sequence is methionine. Position 38 of the reference sequence is tyrosine. (Top SEQ ID NO: 128, bottom SEQ ID NO: 131).

[0080] Re f 4 DTDYITED - GKPVIRIFKKENGEFKIEYDRTFEPY 38

[0081] Tar 4 DTDYITEDQRQRGKPVIRIFKKENGEFKIEYDRTFEPM 42

[0082] In a fourth example, the target sequence differs in sequence at amino position 38, 42, 43, and 44. Position 38 of the target sequence corresponds to position 38 of the reference sequence. Position 38 of the target sequence is methionine. Position 38 of the reference sequence is tyrosine. (Top SEQ ID NO: 132, bottom SEQ ID NO: 133).

[0083] Ref 4 DTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYGGYSTV 44

[0084] Tar 4 DTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPMGGYRSE 44

[0085] In some instances, two different protein sequences may not correspond with one another. For example, this may occur when the sequence identity or structural identity between two protein sequences is so low that an alignment cannot be made.

[0086] “Uracil tolerance” refers to the activity (or relative activity) of a polymerase (e.g., KOD polymerase or a variant thereof) when the polymerase is amplifying a DNA template that comprises one or more uracil. Wildtype KOD polymerase generally does not have detectable uracil tolerance and typically stalls when it encounters uracil on the template. In some embodiments, an increase in uracil tolerance of a KOD polymerase variant is relative to

[0087] #14338400v1 wildtype KOD polymerase (e.g., increased relative to the activity of KOD polymerase having an amino acid sequence of SEQ ID NO: 1).

[0088] In some embodiments, this disclosure provides KOD polymerase variants that increase uracil tolerance of the KOD polymerase. For example, a KOD polymerase variant may increase the activity of KOD polymerase when KOD polymerase is amplifying a polynucleotide that comprises uracil (or an uracil derivative like deoxyuracil and dihydrouracil). In some embodiments, KOD polymerase variants comprising mutations at positions 116, P36, P90 and V93 have increased KOD polymerase uracil tolerance.

[0089] “Salt tolerance” refers to the activity (or relative activity) of a polymerase (e.g., a KOD polymerase or variant thereof) when the polymerase is amplifying a DNA template in a salt condition (e.g., salt concentration or salt type) that is altered relative to typical salt conditions used in a KOD polymerase amplification reaction (e.g., a KOD polymerase PCR). A KOD polymerase variant with increased salt tolerance includes a KOD polymerase variant that has increased activity in altered salt conditions (e.g., increased activity in increased KC1 and / or decreased Mg2+relative to typical salt concentration of KOD polymerase amplification) e.g., compared to a wildtype Taq polymerase. In some embodiments, a KOD polymerase variant described herein has altered Mg2+(e.g., the KOD polymerase variants has higher relative activity than wildtype KOD polymerase in Mg2+concentrations that are higher and / or lower than typically used in a PCR with wildtype KOD polymerase). In some embodiments, a KOD polymerase variant with increased salt tolerance has activity in salt conditions in which wildtype KOD polymerase does not have activity. Typical monovalent cationic salt concentrations e.g. KC1 used in PCR with WT KOD polymerase are between 20 - 150 mM. Typical divalent cation concentrations, generally MgCh, are between 1.5 - 4 mM. Reaction mixes also include a buffering agent e.g. Tris-Cl with a pH between 8 and 9, as well as dNTPs between a concentration of 100 - 300 uM each.

[0090] In some embodiments, a mutation in a KOD polymerase variant at one or more of position E325, S347, F356, L396, 1488, F587, V589, T605, G634, R641, E664, A675, G677, A747, or F748 corresponding to SEQ ID NO: 1 alters the salt tolerance of KOD polymerase.

[0091] E325 KOD Polymerase Variants

[0092] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position E325 of SEQ ID NO: 1.

[0093] In some embodiments, this disclosure provides a KOD polymerase variant comprising a methionine, threonine, lysine, serine, arginine, leucine, proline, histidine, glutamine, valine,

[0094] #14338400v1 alanine, glycine, phenylalanine, cysteine, or tyrosine at a position corresponding to E325 of SEQ ID NO: 1 (e.g., E325M, E325T, E325K, E325S, E325R, E325L, E325P, E325H, E325Q, E325V, E325A, E325G, E325F, E325C, or E325Y in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides a KOD polymerase variant comprising a methionine, threonine, lysine, serine, arginine, leucine, proline, histidine, glutamine, valine, alanine, glycine, phenylalanine, cysteine, or tyrosine at position E325 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine, threonine, lysine, serine, arginine, leucine, proline, histidine, glutamine, valine, alanine, glycine, phenylalanine, cysteine, or tyrosine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine, threonine, lysine, serine, arginine, leucine, proline, histidine, glutamine, valine, alanine, glycine, phenylalanine, cysteine, or tyrosine at position E325 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a methionine, threonine, lysine, serine, arginine, leucine, proline, histidine, glutamine, valine, alanine, glycine, phenylalanine, cysteine, or tyrosine at position E325 of SEQ ID NO: 1.

[0095] In some embodiments, the KOD polymerase variant comprises a methionine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a threonine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a lysine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a serine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an arginine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a leucine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a proline at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a histidine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a glutamine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a valine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an alanine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant

[0096] #14338400v1 comprises a glycine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a phenylalanine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a cysteine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a tyrosine at a position corresponding to E325 of SEQ ID NO: 1.

[0097] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a serine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a leucine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a proline at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a histidine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a valine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an alanine at position E325 of SEQ ID NO: 1. In some embodiments, the

[0098] #14338400v1 KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glycine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a phenylalanine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cysteine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tyrosine at position E325 of SEQ ID NO: 1.

[0099] In some embodiments, the KOD polymerase variant comprises a methionine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a threonine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a lysine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a serine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an arginine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a leucine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a proline at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a histidine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a glutamine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a valine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an alanine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a glycine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a phenylalanine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a cysteine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a tyrosine at position E325 of SEQ ID NO: 1.

[0100] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2-6. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2-6. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 2-6. In some

[0101] #14338400v1 embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 2-6. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 2-6. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NOs: 2-6.

[0102] In some embodiments, this disclosure provides a KOD polymerase variant comprises an asparagine, aspartic acid, isoleucine, or tryptophan at a position corresponding to E325 of SEQ ID NO: 1.

[0103] In some embodiments, this disclosure provides a KOD polymerase variant comprising asparagine, aspartic acid, isoleucine or tryptophan at position E325 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine, aspartic acid, glutamic acid, isoleucine or tryptophan at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine, aspartic acid, glutamic acid, isoleucine or tryptophan at position E325 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises asparagine, aspartic acid, isoleucine or tryptophan at position E325 of SEQ ID NO: 1.

[0104] In some embodiments, the KOD polymerase variant comprises a asparagine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a aspartic acid at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a isoleucine at a position corresponding to E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a tryptophan at a position corresponding to E325 of SEQ ID NO: 1.

[0105] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an isoleucine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least

[0106] #14338400v1 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tryptophan at position E325 of SEQ ID NO: 1.

[0107] In some embodiments, the KOD polymerase variant comprises an asparagine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an aspartic acid at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an isoleucine at position E325 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises a tryptophan at position E325 of SEQ ID NO: 1.

[0108] S347 KOD Polymerase Variants

[0109] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position E347 of SEQ ID NO: 1.

[0110] In some embodiments, a KOD polymerase variant comprises: a threonine, asparagine, arginine, lysine, glutamine, or valine at a position corresponding to S347 of SEQ ID NO: 1 (e.g., S347T, S347N, S347R, S347K, S347Q, or S347V in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, a KOD polymerase variant comprises: a threonine, asparagine, arginine, lysine, glutamine, or valine at position S347 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine, asparagine, arginine, lysine, glutamine, or valine at position S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine, asparagine, arginine, lysine, glutamine, or valine at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a threonine, asparagine, arginine, lysine, glutamine, or valine at position S347 of SEQ ID NO: 1.

[0111] In some embodiments, a KOD polymerase variant comprises a threonine at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an asparagine at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at a position corresponding to S347 of SEQ ID NO: 1. In some

[0112] #14338400v1 embodiments, a KOD polymerase variant comprises a valine at a position corresponding to S347 of SEQ ID NO: 1.

[0113] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine at position S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamine at position S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a valine at position S347 of SEQ ID NO: 1.

[0114] In some embodiments, a KOD polymerase variant comprises a threonine at position S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an asparagine at position S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at position S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at position S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at position S347 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at position S347 of SEQ ID NO: 1.

[0115] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 7-9. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 7-9. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 7-9. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 7-9. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID

[0116] #14338400v1 NOs: 7-9. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NOs: 7-9.

[0117] In some embodiments, this disclosure provides a KOD polymerase variant comprising alanine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, phenylalanine, proline, tryptophan, or tyrosine at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising alanine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, phenylalanine, proline, tryptophan, or tyrosine at position S347 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, phenylalanine, proline, tryptophan, or tyrosine at position S347 of SEQ ID NO: 1.

[0118] In some embodiments, the KOD polymerase variant comprises alanine at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises aspartic acid at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises isoleucine at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at a position corresponding to S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at a position corresponding to S347 of SEQ ID NO: 1.

[0119] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO:

[0120] #14338400v1 1 and alanine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and aspartic acid at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and cysteine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamic acid at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glycine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and histidine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and isoleucine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and leucine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and phenylalanine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and proline at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tryptophan at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tyrosine at position S347 of SEQ ID NO: 1.

[0121] In some embodiments, the KOD polymerase variant comprises alanine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises aspartic acid at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant

[0122] #14338400v1 comprises cysteine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises isoleucine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at position S347 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at position S347 of SEQ ID NO: 1.

[0123] F356 KOD Polymerase Variants

[0124] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position F356 of SEQ ID NO: 1.

[0125] In some embodiments, a KOD polymerase variant comprises: an isoleucine, methionine, asparagine, lysine, serine, arginine, leucine, valine, alanine, aspartic acid, glycine, or cysteine at a position corresponding to F356 of SEQ ID NO: 1 (e.g., F356I, F356M, F356N, F356K, F356S, F356R, F356L, F356V, F356A, F356D, F356G, or F356C in a KOD polymerase corresponding to SEQ ID NO: 1).

[0126] In some embodiments, a KOD polymerase variant comprises: an isoleucine, methionine, asparagine, lysine, serine, arginine, leucine, valine, alanine, aspartic acid, glycine, or cysteine at position F356 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0127] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an isoleucine, methionine, asparagine, lysine, serine, arginine, leucine, valine, alanine, aspartic acid, glycine, or cysteine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an

[0128] #14338400v1 isoleucine, methionine, asparagine, lysine, serine, arginine, leucine, valine, alanine, aspartic acid, glycine, or cysteine at position F356 of SEQ ID NO: 1.

[0129] In some embodiments, a KOD polymerase variant comprises an isoleucine, methionine, asparagine, lysine, serine, arginine, leucine, valine, alanine, aspartic acid, glycine, or cysteine at position F356 of SEQ ID NO: 1.

[0130] In some embodiments, a KOD polymerase variant comprises a isoleucine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a methionine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an asparagine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a serine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a leucine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an aspartic acid at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glycine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a cysteine at a position corresponding to F356 of SEQ ID NO: 1.

[0131] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an isoleucine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with

[0132] #14338400v1 SEQ ID NO: 1 and a serine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a leucine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a valine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a alanine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a aspartic acid at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glycine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cysteine at position F356 of SEQ ID NO: 1.

[0133] In some embodiments, a KOD polymerase variant comprises an isoleucine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a methionine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an asparagine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a serine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a leucine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an aspartic acid at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glycine at position F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a cysteine at position F356 of SEQ ID NO: 1.

[0134] #14338400v1 In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 10. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 10. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 10. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 10. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 10. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 10.

[0135] In some embodiments, this disclosure provides a KOD polymerase variant comprising glutamic acid, glutamine, histidine, proline, threonine, tryptophan, or tyrosine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising glutamic acid, glutamine, histidine, proline, threonine, tryptophan, or tyrosine at position F356 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamic acid, glutamine, histidine, proline, threonine, tryptophan, or tyrosine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamic acid, glutamine, histidine, proline, threonine, tryptophan, or tyrosine at position F356 of SEQ ID NO: 1.

[0136] In some embodiments, the KOD polymerase variant comprises glutamic acid at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at a position corresponding to F356 of SEQ ID NO: 1.

[0137] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO:

[0138] #14338400v1 1 and glutamic acid at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamine at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and histidine at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and proline at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and threonine at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tryptophan at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tyrosine at position F356 of SEQ ID NO: 1.

[0139] In some embodiments, the KOD polymerase variant comprises glutamic acid at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamine at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at position F356 of SEQ ID NO: 1.

[0140] In some embodiments, this disclosure provides a KOD polymerase variant comprising glutamic acid, glutamine, histidine, proline, threonine, tryptophan, or tyrosine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising glutamic acid, glutamine, histidine, proline, threonine, tryptophan, or tyrosine at position F356 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamic acid, glutamine, histidine, proline, threonine, tryptophan, or tyrosine at position F356 of SEQ ID NO: 1.

[0141] #14338400v1 In some embodiments, the KOD polymerase variant comprises glutamic acid at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at a position corresponding to F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at a position corresponding to F356 of SEQ ID NO: 1.

[0142] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamic acid at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamine at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and histidine at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and proline at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and threonine at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tryptophan at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tyrosine at position F356 of SEQ ID NO: 1.

[0143] In some embodiments, the KOD polymerase variant comprises glutamic acid at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamine at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at position F356 of SEQ ID NO: 1. In some

[0144] #14338400v1 embodiments, the KOD polymerase variant comprises tryptophan at position F356 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at position F356 of SEQ ID NO: 1.

[0145] L396 KOD Polymerase Variants

[0146] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position L396 of SEQ ID NO: 1.

[0147] In some embodiments, a KOD polymerase variant comprises: a threonine, lysine, serine, arginine, proline, histidine, glutamine, alanine, aspartic acid, glutamic acid, glycine, tyrosine, or cysteine at a position corresponding to L396 of SEQ ID NO: 1 (e.g., L396T, L396K, L396S, L396R, L396P, L396H, L396Q, L396A, L396D, L396E, L396G, L396Y, or L396C in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, a KOD polymerase variant comprises: a threonine, lysine, serine, arginine, proline, histidine, glutamine, alanine, aspartic acid, glutamic acid, glycine, tyrosine, or cysteine at position L396 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine, lysine, serine, arginine, proline, histidine, glutamine, alanine, aspartic acid, glutamic acid, glycine, tyrosine, or cysteine at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine, lysine, serine, arginine, proline, histidine, glutamine, alanine, aspartic acid, glutamic acid, glycine, tyrosine, or cysteine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a threonine, lysine, serine, arginine, proline, histidine, glutamine, alanine, aspartic acid, glutamic acid, glycine, tyrosine, or cysteine at position L396 of SEQ ID NO: 1.

[0148] In some embodiments, a KOD polymerase variant comprises a threonine at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a serine at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a proline at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at a position corresponding to

[0149] #14338400v1 L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an aspartic acid at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamic acid at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glycine at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a cysteine at a position corresponding to L396 of SEQ ID NO: 1.

[0150] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a serine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a proline at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a histidine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an alanine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%,

[0151] #14338400v1 or at least 99%) identity with SEQ ID NO: 1 and a glutamic acid at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glycine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tyrosine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cysteine at position L396 of SEQ ID NO: 1.

[0152] In some embodiments, a KOD polymerase variant comprises a threonine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a serine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a proline at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a aspartic acid at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamic acid at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glycine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a cysteine at position L396 of SEQ ID NO: 1.

[0153] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 11-23. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 11-23. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 11-23. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 11-23. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID

[0154] #14338400v1 NOs: 11-23. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NOs: 11-23.

[0155] In some embodiments, this disclosure provides a KOD polymerase variant comprising asparagine, isoleucine, methionine, phenylalanine, tryptophan, or valine at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising asparagine, isoleucine, methionine, phenylalanine, tryptophan, or valine at position L396 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine, isoleucine, methionine, phenylalanine, tryptophan, or valine at position L396 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine, isoleucine, methionine, phenylalanine, tryptophan, or valine at a position corresponding to L396 of SEQ ID NO: 1.

[0156] In some embodiments, the KOD polymerase variant comprises asparagine at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises isoleucine at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at a position corresponding to L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises valine at a position corresponding to L396 of SEQ ID NO: 1.

[0157] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine at position L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and isoleucine at position L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine at position L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and phenylalanine at position L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an

[0158] #14338400v1 amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tryptophan at position L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and valine at position L396 of SEQ ID NO: 1.

[0159] In some embodiments, the KOD polymerase variant comprises asparagine at position L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises isoleucine at position L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at position L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at position L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at position L396 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises valine at position L396 of SEQ ID NO: 1.

[0160] 1488 KOD Polymerase Variants

[0161] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position 1488 of SEQ ID NO: 1.

[0162] In some embodiments, a KOD polymerase variant comprises: an arginine, asparagine, lysine, glutamine, alanine, aspartic acid, or glycine at a position corresponding to 1488 of SEQ ID NO: 1 (e.g., I488R, I488N, I488K, I488Q, I488A, I488D, or I488G in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, a KOD polymerase variant comprises: an arginine, asparagine, lysine, glutamine, alanine, aspartic acid, or glycine at position 1488 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine, asparagine, lysine, glutamine, alanine, aspartic acid, or glycine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine, asparagine, lysine, glutamine, alanine, aspartic acid, or glycine at position 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine, asparagine, lysine, glutamine, alanine, aspartic acid, or glycine at position 1488 of SEQ ID NO: 1.

[0163] In some embodiments, a KOD polymerase variant comprises an arginine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant

[0164] #14338400v1 comprises an asparagine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an aspartic acid at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an glycine at a position corresponding to 1488 of SEQ ID NO: 1.

[0165] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamine at position 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an alanine at position 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid at position 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glycine at position 1488 of SEQ ID NO: 1.

[0166] In some embodiments, a KOD polymerase variant comprises an arginine at position 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an asparagine at position 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at position 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at position 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at position 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an aspartic acid at position 1488 of SEQ ID

[0167] #14338400v1 NO: 1. In some embodiments, a KOD polymerase variant comprises a glycine at position 1488 of SEQ ID NO: 1.

[0168] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 24. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 24. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 24. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 24. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 24. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 24.

[0169] In some embodiments, this disclosure provides a KOD polymerase variant comprising cysteine, glutamic acid, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising cysteine, glutamic acid, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine at position 1488 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and cysteine, glutamic acid, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and cysteine, glutamic acid, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine at position 1488 of SEQ ID NO: 1.

[0170] In some embodiments, the KOD polymerase variant comprises cysteine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at a position

[0171] #14338400v1 corresponding to 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at a position corresponding to 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises valine at a position corresponding to 1488 of SEQ ID NO: 1.

[0172] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and cysteine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamic acid at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and histidine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and leucine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and phenylalanine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and proline at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and serine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and threonine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tryptophan at position 1488 of SEQ ID NO: 1.

[0173] #14338400v1 In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tyrosine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and valine at position 1488 of SEQ ID NO: 1.

[0174] In some embodiments, the KOD polymerase variant comprises cysteine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at position 1488 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises valine at position 1488 of SEQ ID NO: 1.

[0175] F587 KOD Polymerase Variants

[0176] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position F587 of SEQ ID NO: 1.

[0177] In some embodiments, a KOD polymerase variant comprises methionine, asparagine, lysine, histidine, glutamine, tyrosine, cysteine, or glycine at a position corresponding to F587 of SEQ ID NO: 1 (e.g., F587M, F587N, F587K, F587H, F587Q, F587Y, F587C, or F587G in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, a KOD polymerase variant comprises: methionine, asparagine, lysine, histidine, glutamine, tyrosine, cysteine, or glycine at position F587 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine, asparagine, lysine, histidine, glutamine, tyrosine, cysteine, or glycine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD

[0178] #14338400v1 polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine, asparagine, lysine, histidine, glutamine, tyrosine, cysteine, or glycine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises methionine, asparagine, lysine, histidine, glutamine, tyrosine, cysteine, or glycine at position F587 of SEQ ID NO: 1.

[0179] In some embodiments, a KOD polymerase variant comprises an isoleucine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a methionine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an asparagine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a leucine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a cysteine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glycine at a position corresponding to F587 of SEQ ID NO: 1.

[0180] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an isoleucine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a leucine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at

[0181] #14338400v1 least 98%, or at least 99%) identity with SEQ ID NO: 1 and a histidine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a valine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tyrosine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cysteine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glycine at position F587 of SEQ ID NO: 1.

[0182] In some embodiments, a KOD polymerase variant comprises an isoleucine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a methionine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an asparagine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a leucine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a cysteine at position F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glycine at position F587 of SEQ ID NO: 1.

[0183] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 25-34. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 25-34. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 25-34. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least

[0184] #14338400v1 99% identity to any one of SEQ ID NOs: 25-34. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 25-34. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NOs: 25-34.

[0185] In some embodiments, this disclosure provides a KOD polymerase variant comprising alanine, arginine, aspartic acid, glutamic acid, proline, serine, tryptophan at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising alanine, arginine, aspartic acid, glutamic acid, proline, serine, tryptophan at position F587 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine, arginine, aspartic acid, glutamic acid, proline, serine, tryptophan at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine, arginine, aspartic acid, glutamic acid, proline, serine, tryptophan at position F587 of SEQ ID NO: 1.

[0186] In some embodiments, the KOD polymerase variant comprises alanine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises arginine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises aspartic acid at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at a position corresponding to F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at a position corresponding to F587 of SEQ ID NO: 1.

[0187] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine at position F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and arginine at position F587 of SEQ ID NO: 1. In

[0188] #14338400v1 some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and aspartic acid at position F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamic acid at position F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and proline at position F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and serine at position F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and threonine at position F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tryptophan at position F587 of SEQ ID NO: 1.

[0189] In some embodiments, the KOD polymerase variant comprises alanine at position F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises arginine at position F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises aspartic acid at position F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at position F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at position F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at position F587 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at position [R] of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at position F587 of SEQ ID NO: 1.

[0190] V589 KOD Polymerase Variants

[0191] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position V589 of SEQ ID NO: 1.

[0192] In some embodiments, a KOD polymerase variant comprises asparagine, arginine, alanine, glycine, phenylalanine, tyrosine, or tryptophan at a position corresponding to V589 of SEQ ID NO: 1 (e.g., V589N, V589R, V589A, V589G, V589F, V589Y, or V589W in a KOD polymerase corresponding to SEQ ID NO: 1).

[0193] #14338400v1 In some embodiments, a KOD polymerase variant comprises: asparagine, arginine, alanine, glycine, phenylalanine, tyrosine, or tryptophan at position V589 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine, arginine, alanine, glycine, phenylalanine, tyrosine, or tryptophan at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine, arginine, alanine, glycine, phenylalanine, tyrosine, or tryptophan at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises asparagine, arginine, alanine, glycine, phenylalanine, tyrosine, or tryptophan at position V589 of SEQ ID NO: 1.

[0194] In some embodiments, a KOD polymerase variant comprises an asparagine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glycine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at a position corresponding to V589 of SEQ ID NO: 1.

[0195] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a histidine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%,

[0196] #14338400v1 or at least 99%) identity with SEQ ID NO: 1 and a glutamine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an alanine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glycine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a phenylalanine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tyrosine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tryptophan at position V589 of SEQ ID NO: 1.

[0197] In some embodiments, a KOD polymerase variant comprises an asparagine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glycine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at position V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at position V589 of SEQ ID NO: 1.

[0198] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 35-43. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 35-43. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 35-43. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 35-43. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID

[0199] #14338400v1 NOs: 35-43. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 35-43.

[0200] In some embodiments, this disclosure provides a KOD polymerase variant comprising aspartic acid, cysteine, glutamic acid, isoleucine, leucine, lysine, methionine, proline, serine, threonine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising aspartic acid, cysteine, glutamic acid, isoleucine, leucine, lysine, methionine, proline, serine, threonine at position V589 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and aspartic acid, cysteine, glutamic acid, isoleucine, leucine, lysine, methionine, proline, serine, threonine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and aspartic acid, cysteine, glutamic acid, isoleucine, leucine, lysine, methionine, proline, serine, threonine at position V589 of SEQ ID NO: 1.

[0201] In some embodiments, the KOD polymerase variant comprises aspartic acid at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises isoleucine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises lysine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at a position corresponding to V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at a position corresponding to V589 of SEQ ID NO: 1.

[0202] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and aspartic acid at position V589 of SEQ ID NO: 1. In some embodiments, the KOD

[0203] #14338400v1 polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and cysteine at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamic acid at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and isoleucine at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and leucine at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and lysine at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and proline at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and serine at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and threonine at position V589 of SEQ ID NO: 1.

[0204] In some embodiments, the KOD polymerase variant comprises aspartic acid at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises isoleucine at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises lysine at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at position V589 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at position V589 of SEQ ID NO: 1.

[0205] #14338400v1 T605 KOD Polymerase Variants

[0206] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position T605 of SEQ ID NO: 1.

[0207] In some embodiments, a KOD polymerase variant comprises isoleucine, proline, histidine, valine, alanine, phenylalanine, tyrosine, cysteine, or tryptophan at a position corresponding to T605 of SEQ ID NO: 1 (e.g., T605I, T605P, T605H, T605V, T605A, T605F, T605Y, T605C, T605W in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, a KOD polymerase variant comprises: isoleucine, proline, histidine, valine, alanine, phenylalanine, tyrosine, cysteine, or tryptophan at position T605 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and isoleucine, proline, histidine, valine, alanine, phenylalanine, tyrosine, cysteine, or tryptophan at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and isoleucine, proline, histidine, valine, alanine, phenylalanine, tyrosine, cysteine, or tryptophan at position T605 of SEQ ID NO: 1.

[0208] In some embodiments, a KOD polymerase variant comprises isoleucine, proline, histidine, valine, alanine, phenylalanine, tyrosine, cysteine, or tryptophan at position T605 of SEQ ID NO: 1.

[0209] In some embodiments, a KOD polymerase variant comprises an isoleucine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a proline at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a cysteine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at a position corresponding to T605 of SEQ ID NO: 1.

[0210] #14338400v1 In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an isoleucine at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a proline at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a histidine at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a valine at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an alanine at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a phenylalanine at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tyrosine at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cysteine at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tryptophan at position T605 of SEQ ID NO: 1.

[0211] In some embodiments, a KOD polymerase variant comprises an isoleucine at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a proline at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a cysteine at position T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at position T605 of SEQ ID NO: 1.

[0212] #14338400v1 In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 44-52. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 44-52. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 44-52. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 44-52. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 44-52. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NOs: 44-52.

[0213] In some embodiments, this disclosure provides a KOD polymerase variant comprising arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, leucine, lysine, methionine, or serine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, leucine, lysine, methionine, or serine at position T605 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, leucine, lysine, methionine, or serine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, leucine, lysine, methionine, or serine at position T605 of SEQ ID NO: 1.

[0214] In some embodiments, the KOD polymerase variant comprises arginine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises asparagine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises aspartic acid at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments,

[0215] #14338400v1 the KOD polymerase variant comprises lysine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at a position corresponding to T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at a position corresponding to T605 of SEQ ID NO: 1.

[0216] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and arginine at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and aspartic acid at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamic acid at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamine at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glycine at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and leucine at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and lysine at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and serine at position T605 of SEQ ID NO: 1.

[0217] In some embodiments, the KOD polymerase variant comprises arginine at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises asparagine at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises aspartic acid at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at position T605 of SEQ ID NO: 1. In some embodiments, the

[0218] #14338400v1 KOD polymerase variant comprises glutamine at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises lysine at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at position T605 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at position T605 of SEQ ID NO: 1.

[0219] G634 KOD Polymerase Variants

[0220] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position G634 of SEQ ID NO: 1.

[0221] In some embodiments, a KOD polymerase variant comprises: asparagine, lysine, arginine, leucine, proline, glutamine, aspartic acid, glutamic acid, phenylalanine, tyrosine, cysteine, tryptophan, isoleucine, threonine, serine, histidine, valine, or alanine at a position corresponding to G634 of SEQ ID NO: 1 (e.g., G634N, G634K, G634R, G634L, G634P, G634Q, G634D, G634E, G634F, G634Y, G634C, G634W, G634I, G634T, G634S, G634H, G634C, or G634A in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, a KOD polymerase variant comprises: asparagine, lysine, arginine, leucine, proline, glutamine, aspartic acid, glutamic acid, phenylalanine, tyrosine, cysteine, tryptophan, isoleucine, threonine, serine, histidine, valine, or alanine at position G634 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0222] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine, lysine, arginine, leucine, proline, glutamine, aspartic acid, glutamic acid, phenylalanine, tyrosine, cysteine, tryptophan, isoleucine, threonine, serine, histidine, valine, or alanine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine, lysine, arginine, leucine, proline, glutamine, aspartic acid, glutamic acid, phenylalanine, tyrosine, cysteine, tryptophan, isoleucine, threonine, serine, histidine, valine, or alanine at position G634 of SEQ ID NO: 1.

[0223] In some embodiments, a KOD polymerase variant comprises asparagine, lysine, arginine, leucine, proline, glutamine, aspartic acid, glutamic acid, phenylalanine, tyrosine, cysteine, tryptophan, isoleucine, threonine, serine, histidine, valine, or alanine at position G634 of SEQ ID NO: 1.

[0224] #14338400v1 In some embodiments, a KOD polymerase variant comprises an asparagine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a leucine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a proline at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an aspartic acid at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamic acid at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a cysteine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an isoleucine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a threonine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a serine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at a position corresponding to G634 of SEQ ID NO: 1.

[0225] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at

[0226] #14338400v1 least 98%, or at least 99%) identity with SEQ ID NO: 1 and a leucine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a proline at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamic acid at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a phenylalanine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tyrosine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cysteine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tryptophan at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an isoleucine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a serine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a histidine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a valine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant

[0227] #14338400v1 comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an alanine at position G634 of SEQ ID NO: 1.

[0228] In some embodiments, a KOD polymerase variant comprises an asparagine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a leucine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a proline at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an aspartic acid at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamic acid at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a cysteine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an isoleucine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a threonine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a serine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at position G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at position G634 of SEQ ID NO: 1.

[0229] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 53-70. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 53-70. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 53-70. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 53-70. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 53-70. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 53-70.

[0230] #14338400v1 In some embodiments, this disclosure provides a KOD polymerase variant comprising methionine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising methionine at position G634 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine at a position corresponding to G634 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine at position G634 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at a position corresponding to G634 of SEQ ID NO: 1.

[0231] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine at position G634 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at position G634 of SEQ ID NO: 1.

[0232] R641 KOD Polymerase Variants

[0233] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position R641 of SEQ ID NO: 1.

[0234] In some embodiments, a KOD polymerase variant comprises proline, glutamic acid, or alanine at a position corresponding to R641 of SEQ ID NO: 1 (e.g., R641P, R641E, or R641A in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, a KOD polymerase variant comprises: proline, glutamic acid, or alanine at position R641 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and proline, glutamic acid, or alanine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and proline, glutamic acid, or alanine at position R641 of SEQ ID NO: 1.

[0235] In some embodiments, a KOD polymerase variant comprises proline, glutamic acid, or alanine at position R641 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a proline at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamic acid at a position corresponding

[0236] #14338400v1 to R641 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at a position corresponding to R641 of SEQ ID NO: 1.

[0237] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a proline at position R641 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamic acid at position R641 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an alanine at position R641 of SEQ ID NO: 1.

[0238] In some embodiments, a KOD polymerase variant comprises a proline at position R641 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamic acid at position R641 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at position R641 of SEQ ID NO: 1.

[0239] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 71-72. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 71-72. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 71-72. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 71-72. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 71-72. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NOs: 71-72.

[0240] In some embodiments, this disclosure provides a KOD polymerase variant comprising asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine at position R641 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine, aspartic acid, cysteine, glutamine,

[0241] #14338400v1 glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine at position R641 of SEQ ID NO: 1.

[0242] In some embodiments, the KOD polymerase variant comprises asparagine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises aspartic acid at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises isoleucine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises lysine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at a position corresponding to R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises valine at a position corresponding to R641 of SEQ ID NO: 1.

[0243] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at

[0244] #14338400v1 least 98%, or at least 99%) identity with SEQ ID NO: 1 and aspartic acid at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and cysteine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glycine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and histidine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and isoleucine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and leucine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and lysine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and phenylalanine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and serine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and threonine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tryptophan at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tyrosine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD

[0245] #14338400v1 polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and valine at position R641 of SEQ ID NO: 1.

[0246] In some embodiments, the KOD polymerase variant comprises asparagine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises aspartic acid at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises isoleucine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises lysine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at position R641 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises valine at position R641 of SEQ ID NO: 1.

[0247] E664 KOD Polymerase Variants

[0248] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position E664 of SEQ ID NO: 1.

[0249] In some embodiments, a KOD polymerase variant comprises: a methionine, threonine, asparagine, serine, arginine, valine, or isoleucine at a position corresponding to E664 of SEQ ID NO: 1 (e.g., E664M, E664T, E664N, E664S, E664R, E664Q, E664V, E664I, or in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, a KOD polymerase variant comprises: a methionine, threonine, asparagine, serine, arginine, valine, or isoleucine at position E664 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at

[0250] #14338400v1 least 99%) identity with SEQ ID NO: 1 and a methionine, threonine, asparagine, serine, arginine, valine or isoleucine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine, threonine, asparagine, serine, arginine, valine or isoleucine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a methionine, threonine, asparagine, serine, arginine, valine or isoleucine at position E664 of SEQ ID NO: 1.

[0251] In some embodiments, a KOD polymerase variant comprises a methionine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a threonine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an asparagine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a serine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an isoleucine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a cysteine at a position corresponding to E664 of SEQ ID NO: 1.

[0252] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a serine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase

[0253] #14338400v1 variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a valine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an isoleucine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cysteine at position E664 of SEQ ID NO: 1.

[0254] In some embodiments, a KOD polymerase variant comprises a methionine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a threonine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an asparagine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a serine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an isoleucine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a cysteine at position E664 of SEQ ID NO: 1.

[0255] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 73-80. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 73-80. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 73-80. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 73-80. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 73-80. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NOs: 73-80.

[0256] #14338400v1 In some embodiments, this disclosure provides a KOD polymerase variant comprising alanine, aspartic acid, glycine, histidine, leucine, proline, tryptophan, or tyrosine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising alanine, aspartic acid, glycine, histidine, leucine, proline, tryptophan, or tyrosine at position E664 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine, aspartic acid, glycine, histidine, leucine, proline, tryptophan, or tyrosine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine, aspartic acid, glycine, histidine, leucine, proline, tryptophan, or tyrosine at position E664 of SEQ ID NO: 1.

[0257] In some embodiments, the KOD polymerase variant comprises alanine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises aspartic acid at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at a position corresponding to E664 of SEQ ID NO: 1.

[0258] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and aspartic acid at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glycine at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase

[0259] #14338400v1 variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and histidine at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and leucine at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and phenylalanine at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and proline at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tryptophan at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tyrosine at position E664 of SEQ ID NO: 1.

[0260] In some embodiments, the KOD polymerase variant comprises alanine at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises aspartic acid at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at position E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at position E664 of SEQ ID NO: 1.

[0261] A675 KOD Polymerase Variants

[0262] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position A675 of SEQ ID NO: 1.

[0263] In some embodiments, a KOD polymerase variant comprises isoleucine, threonine, lysine, arginine, histidine, valine, or tryptophan at a position corresponding to A675 of SEQ ID NO: 1 (e.g., A675I, A675M, A675T, A675K, A675R, A675H, A675V, A675W) in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, a KOD polymerase

[0264] #14338400v1 variant comprises isoleucine, threonine, lysine, arginine, histidine, valine, or tryptophan at position A675 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and isoleucine, threonine, lysine, arginine, histidine, valine, or tryptophan at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and isoleucine, threonine, lysine, arginine, histidine, valine, or tryptophan at position A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises isoleucine, threonine, lysine, arginine, histidine, valine, or tryptophan at position A675 of SEQ ID NO: 1.

[0265] In some embodiments, a KOD polymerase variant comprises a isoleucine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a methionine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a threonine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at a position corresponding to A675 of SEQ ID NO: 1.

[0266] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an isoleucine at position A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine at position A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine at position A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid

[0267] #14338400v1 sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a histidine at position A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a valine at position A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tryptophan at position A675 of SEQ ID NO: 1.

[0268] In some embodiments, a KOD polymerase variant comprises a isoleucine at position A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a methionine at position A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a threonine at position A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at position A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at position A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at position A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at position A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at position A675 of SEQ ID NO: 1.

[0269] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 81-88. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 81-88. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 81-88. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 81-88. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 81-88. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NOs: 81-88.

[0270] In some embodiments, this disclosure provides a KOD polymerase variant comprising asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, leucine, proline, serine, tyrosine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising asparagine, aspartic acid, cysteine,

[0271] #14338400v1 glutamic acid, glutamine, glycine, leucine, proline, serine, tyrosine at position A675 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, leucine, proline, serine, tyrosine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, leucine, proline, serine, tyrosine at position A675 of SEQ ID NO: 1.

[0272] In some embodiments, the KOD polymerase variant comprises asparagine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises aspartic acid at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at a position corresponding to A675 of SEQ ID NO: 1.

[0273] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and aspartic acid at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and cysteine at position A675 of SEQ ID NO: 1. In some embodiments, the KOD

[0274] #14338400v1 polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamic acid at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamine at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glycine at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and leucine at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and phenylalanine at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and proline at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and serine at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tyrosine at position A675 of SEQ ID NO: 1.

[0275] In some embodiments, the KOD polymerase variant comprises asparagine at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises aspartic acid at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamine at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at position A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at position A675 of SEQ ID NO: 1.

[0276] #14338400v1 G677 KOD Polymerase Variants

[0277] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position G677 of SEQ ID NO: 1.

[0278] In some embodiments, a KOD polymerase variant comprises: a methionine, threonine, serine, arginine, leucine, proline, histidine, glutamine, phenylalanine, lysine, or alanine at a position corresponding to G677 of SEQ ID NO: 1 (e.g., G677M, G677T, G677S, G677R, G677L, G677P, G677H, G677Q, G677K, or G677A) in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, a KOD polymerase variant comprises: a methionine, threonine, serine, arginine, leucine, proline, histidine, glutamine, phenylalanine, lysine, or alanine at position G677 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine, threonine, serine, arginine, leucine, proline, histidine, glutamine, phenylalanine, lysine, or alanine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine, threonine, serine, arginine, leucine, proline, histidine, glutamine, phenylalanine, lysine, or alanine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a methionine, threonine, serine, arginine, leucine, proline, histidine, glutamine, phenylalanine, lysine, or alanine at position G677 of SEQ ID NO: 1.

[0279] In some embodiments, a KOD polymerase variant comprises a methionine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a threonine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a serine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a leucine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a proline at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, a

[0280] #14338400v1 KOD polymerase variant comprises a lysine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at a position corresponding to G677 of SEQ ID NO: 1.

[0281] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a serine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a leucine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a proline at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a histidine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a phenylalanine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an alanine at position G677 of SEQ ID NO: 1.

[0282] In some embodiments, a KOD polymerase variant comprises a methionine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a threonine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant

[0283] #14338400v1 comprises a serine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a leucine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a proline at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at position G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an alanine at position G677 of SEQ ID NO: 1.

[0284] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 89-97. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 89-97. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 89-97. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 89-97. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 89-97. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NOs: 89-97.

[0285] In some embodiments, this disclosure provides a KOD polymerase variant comprising asparagine, aspartic acid, cysteine, glutamic acid, isoleucine, tryptophan, tyrosine, or valine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising asparagine, aspartic acid, cysteine, glutamic acid, isoleucine, tryptophan, tyrosine, or valine at position G677 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine, aspartic acid, cysteine, glutamic acid, isoleucine, tryptophan, tyrosine, or valine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine, aspartic acid, cysteine, glutamic acid, isoleucine, tryptophan, tyrosine, or valine at position G677 of SEQ ID NO: 1.

[0286] #14338400v1 In some embodiments, the KOD polymerase variant comprises asparagine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises aspartic acid at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises isoleucine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at a position corresponding to G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises valine at a position corresponding to G677 of SEQ ID NO: 1.

[0287] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine at position G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and aspartic acid at position G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and cysteine at position G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamic acid at position G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and isoleucine at position G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tryptophan at position G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tyrosine at position G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and valine at position G677 of SEQ ID NO: 1.

[0288] #14338400v1 In some embodiments, the KOD polymerase variant comprises asparagine at position G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises aspartic acid at position G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at position G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at position G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises isoleucine at position G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at position G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at position G677 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises valine at position G677 of SEQ ID NO: 1.

[0289] A747 KOD Polymerase Variants

[0290] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position A747 of SEQ ID NO: 1.

[0291] In some embodiments, a KOD polymerase variant comprises: methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at a position corresponding to A747 of SEQ ID NO: 1 (e.g., A747M, A747N, A747L, A747H, A747D, A747F, A747Y, A747W, A747I, and A747E in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, a KOD polymerase variant comprises: methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1.

[0292] #14338400v1 In some embodiments, a KOD polymerase variant comprises a methionine at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an asparagine at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a leucine at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an isoleucine at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamic acid at a position corresponding to A747 of SEQ ID NO: 1.

[0293] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a leucine at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a histidine at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a phenylalanine at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tyrosine at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least

[0294] #14338400v1 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tryptophan at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an isoleucine at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamic acid at position A747 of SEQ ID NO: 1.

[0295] In some embodiments, a KOD polymerase variant comprises a methionine at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an asparagine at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a leucine at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a aspartic acid at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an isoleucine at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamic acid at position A747 of SEQ ID NO: 1.

[0296] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 98-105. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 98-105. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 98-105. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 98-105. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 98-105. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NOs: 98-105.

[0297] In some embodiments, this disclosure provides a KOD polymerase variant comprising arginine, cysteine, glutamine, glycine, lysine, proline, serine, threonine, or valine at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising arginine, cysteine, glutamine, glycine, lysine, proline,

[0298] #14338400v1 serine, threonine, or valine at position A747 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and arginine, cysteine, glutamine, glycine, lysine, proline, serine, threonine, or valine at position A747 of SEQ ID NO: 1.

[0299] In some embodiments, the KOD polymerase variant comprises arginine at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamine at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises lysine at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises valine at a position corresponding to A747 of SEQ ID NO: 1.

[0300] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and arginine at position A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and cysteine at position A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamine at position A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glycine at position A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and lysine at position A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and proline at position A747 of SEQ ID NO: 1. In some

[0301] #14338400v1 embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and serine at position A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and threonine at position A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and valine at position A747 of SEQ ID NO: 1.

[0302] In some embodiments, the KOD polymerase variant comprises arginine at position A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at position A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamine at position A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at position A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises lysine at position A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at position A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at position A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at position A747 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises valine at position A747 of SEQ ID NO: 1.

[0303] F748 KOD Polymerase Variants

[0304] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position F748 of SEQ ID NO: 1.

[0305] In some embodiments, a KOD polymerase variant comprises lysine, arginine, histidine, aspartic acid, tyrosine, tryptophan, or serine at a position corresponding to F748 of SEQ ID NO: 1 (e.g., F748K, F748R, F748H, F748D, F748Y, F748W, or F748S in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, a KOD polymerase variant comprises: lysine, arginine, histidine, aspartic acid, tyrosine, tryptophan, or serine at position F748 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and lysine, arginine, histidine, aspartic acid, tyrosine, tryptophan, or serine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%,

[0306] #14338400v1 or at least 99%) identity with SEQ ID NO: 1 and lysine, arginine, histidine, aspartic acid, tyrosine, tryptophan, or serine at position F748 of SEQ ID NO: 1.

[0307] In some embodiments, a KOD polymerase variant comprises lysine, arginine, histidine, aspartic acid, tyrosine, tryptophan, or serine at position F748 of SEQ ID NO: 1.

[0308] In some embodiments, a KOD polymerase variant comprises a lysine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an aspartic acid at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a serine at a position corresponding to F748 of SEQ ID NO: 1.

[0309] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a histidine at position F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid at position F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tyrosine at position F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tryptophan at position F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a serine at position F748 of SEQ ID NO: 1.

[0310] In some embodiments, a KOD polymerase variant comprises a lysine at position F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at

[0311] #14338400v1 position F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at position F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an aspartic acid at position F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at position F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at position F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a serine at position F748 of SEQ ID NO: 1.

[0312] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 106-111. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 106-111. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 106-111. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 106-111. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 106-111. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NOs: 106-111.

[0313] In some embodiments, this disclosure provides a KOD polymerase variant comprising alanine, asparagine, cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine, methionine, proline, threonine, valine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising alanine, asparagine, cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine, methionine, proline, threonine, or valine at position F748 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine, asparagine, cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine, methionine, proline, threonine, or valine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine, asparagine, cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine, methionine, proline, threonine, or valine at position F748 of SEQ ID NO: 1.

[0314] In some embodiments, the KOD polymerase variant comprises alanine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant

[0315] #14338400v1 comprises asparagine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises isoleucine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at a position corresponding to F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises valine at a position corresponding to F748 of SEQ ID NO: 1.

[0316] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and cysteine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamic acid at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glycine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and isoleucine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%,

[0317] #14338400v1 or at least 99%) identity with SEQ ID NO: 1 and leucine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and proline at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and threonine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and valine at position F748 of SEQ ID NO: 1.

[0318] In some embodiments, the KOD polymerase variant comprises alanine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises asparagine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises isoleucine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises leucine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises methionine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at position F748 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises valine at position F748 of SEQ ID NO: 1.

[0319] E664 and A747 KOD Polymerase Variants

[0320] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position E664 and a mutation corresponding to A747 of SEQ ID NO: 1.

[0321] In some embodiments, a KOD polymerase variant comprises: a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at a position corresponding to E664 of SEQ ID NO: 1 and a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant

[0322] #14338400v1 comprises: a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at position E664 of SEQ ID NO: 1; a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1; and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0323] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1; a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at position E664 of SEQ ID NO: 1; and a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1.

[0324] In some embodiments, a KOD polymerase variant comprises: a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at position E664 of SEQ ID NO: 1; and a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1.

[0325] In some embodiments, a KOD polymerase variant comprises: a lysine at a position corresponding to E664 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: a lysine at position E664 and an aspartic acid at position A747 of SEQ ID NO: 1, and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position E664 and an aspartic acid at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at position E664 and an aspartic acid at position A747 of SEQ ID NO: 1.

[0326] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 112. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 112. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 112. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 112. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 112. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 112.

[0327] #14338400v1 T605 and E664 KOD Polymerase Variants

[0328] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position T605 and a mutation corresponding to E664of SEQ ID NO: 1.

[0329] In some embodiments, a KOD polymerase variant comprises isoleucine, proline, histidine, valine, alanine, phenylalanine, tyrosine, cysteine, or tryptophan at a position corresponding to T605 of SEQ ID NO: 1 and a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at a position corresponding to E664 of SEQ ID NO: 1.

[0330] In some embodiments, a KOD polymerase variant comprises isoleucine, proline, histidine, valine, alanine, phenylalanine, tyrosine, cysteine, or tryptophan at position T605 of SEQ ID NO: 1 and a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at position E664 (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0331] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1; isoleucine, proline, histidine, valine, alanine, phenylalanine, tyrosine, cysteine, or tryptophan at position T605 of SEQ ID NO: 1; and a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at position E664 in SEQ ID NO: 1.

[0332] In some embodiments, a KOD polymerase variant comprises: a tyrosine at a position corresponding to T605 and a lysine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: a tyrosine at position T605 and a lysine at position E664 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tyrosine at position T605 and a lysine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine corresponding to T605 and a lysine corresponding to E664 of SEQ ID NO: 1.

[0333] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 113. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 113. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 113. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 113. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least

[0334] #14338400v1 99.5% identity to SEQ ID NO: 113. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 113.

[0335] F587 and E664 KOD Polymerase Variants

[0336] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position F587 and a mutation corresponding to E664 of SEQ ID NO: 1.

[0337] In some embodiments, a KOD polymerase variant comprises isoleucine, methionine, asparagine, lysine, leucine, histidine, glutamine, valine, tyrosine, cysteine, or glycine at position F587 of SEQ ID NO: 1, and a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at position E664 of SEQ ID NO: 1.

[0338] In some embodiments, a KOD polymerase variant comprises: isoleucine, methionine, asparagine, lysine, leucine, histidine, glutamine, valine, tyrosine, cysteine, or glycine at position F587 of SEQ ID NO: 1; a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at a position corresponding to E664 of SEQ ID NO: 1, and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0339] In some embodiments, a KOD polymerase variant comprises: isoleucine, methionine, asparagine, lysine, leucine, histidine, glutamine, valine, tyrosine, cysteine, or glycine at position F587 of SEQ ID NO: 1; and a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at a position corresponding to E664 of SEQ ID NO: 1.

[0340] In some embodiments, a KOD polymerase variant comprises isoleucine, methionine, asparagine, lysine, leucine, histidine, glutamine, valine, tyrosine, cysteine, or glycine at position F587 of SEQ ID NO: 1; and a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at a position corresponding to E664 of SEQ ID NO: 1.

[0341] In some embodiments, a KOD polymerase variant comprises: a methionine at a position corresponding to F587 and a lysine at a position corresponding to E664 of SEQ ID NO: 1.

[0342] In some embodiments, a KOD polymerase variant comprises a methionine at position F587 and a lysine at position E664 of SEQ ID NO: 1, and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0343] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine at position F587 and a lysine at position E664 of SEQ ID NO: 1.

[0344] In some embodiments, a KOD polymerase variant comprises a methionine at position F587 and a lysine at position E664 of SEQ ID NO: 1.

[0345] #14338400v1 In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 114. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 114. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 114. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 114. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 114. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 114.

[0346] S347 and E664 KOD Polymerase Variants

[0347] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position S347 and a mutation corresponding to E664 of SEQ ID NO: 1.

[0348] In some embodiments, a KOD polymerase variant comprises: a threonine, asparagine, arginine, lysine, glutamine, or valine at a position corresponding to S347 of SEQ ID NO: 1; and a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: a threonine, asparagine, arginine, lysine, glutamine, or valine at position S347 of SEQ ID NO: 1; a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at position E664 of SEQ ID NO: 1; and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1; a threonine, asparagine, arginine, lysine, glutamine, or valine at position S347 of SEQ ID NO: 1; and a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a threonine, asparagine, arginine, lysine, glutamine, or valine at position S347 of SEQ ID NO: 1; and a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at position E664 of SEQ ID NO: 1.

[0349] In some embodiments, a KOD polymerase variant comprises an arginine at a position corresponding to S347 and a lysine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an arginine at position S347; a lysine at position E664 of SEQ ID NO: 1; and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant

[0350] #14338400v1 comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1; an arginine at position S347; and a lysine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at position S347 and a lysine at position E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a methionine at position F587 and a lysine at position E664 of SEQ ID NO: 1.

[0351] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 115. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 115. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 115. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 115. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 115. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 115.

[0352] G677 and A747 KOD Polymerase Variant

[0353] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position G677 and a mutation corresponding to A747 of SEQ ID NO: 1.

[0354] In some embodiments, a KOD polymerase variant comprises: a methionine, threonine, serine, arginine, leucine, proline, histidine, glutamine, phenylalanine, lysine, or alanine at a position corresponding to G677 of SEQ ID NO: 1; and a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at a position corresponding to A747 of SEQ ID NO: 1.

[0355] In some embodiments, a KOD polymerase variant comprises: a methionine, threonine, serine, arginine, leucine, proline, histidine, glutamine, phenylalanine, lysine, or alanine at position G677 of SEQ ID NO: 1; a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1; and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0356] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine, threonine, serine, arginine, leucine, proline, histidine, glutamine, phenylalanine, lysine, or alanine at position G677 of SEQ ID NO: 1; and a methionine,

[0357] #14338400v1 asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1.

[0358] In some embodiments, a KOD polymerase variant comprises a methionine, threonine, serine, arginine, leucine, proline, histidine, glutamine, phenylalanine, lysine, or alanine at position G677 of SEQ ID NO: 1; and a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1.

[0359] In some embodiments, a KOD polymerase variant comprises: a proline at a position corresponding to G677 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: a proline at position G677 and an aspartic acid at position A747 of SEQ ID NO: 1; and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1; and a proline at position G677 and an aspartic acid at position A747 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a proline at position G677 and an aspartic acid at position A747 of SEQ ID NO: 1.

[0360] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 116. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 116. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 116. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 116. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 116. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 116.

[0361] A675 and A747 KOD Polymerase Variant

[0362] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position A675 and a mutation corresponding to A747 of SEQ ID NO: 1.

[0363] In some embodiments, a KOD polymerase variant comprises: an isoleucine, methionine, threonine, lysine, arginine, histidine, valine, or tryptophan at a position corresponding to A675 of SEQ ID NO: 1; and a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine,

[0364] #14338400v1 tyrosine, tryptophan, isoleucine, or glutamic acid at a position corresponding to A747 of SEQ ID

[0365] NO: 1.

[0366] In some embodiments, a KOD polymerase variant comprises: an isoleucine, methionine, threonine, lysine, arginine, histidine, valine, or tryptophan at position A675 of SEQ ID NO: 1; and a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1.

[0367] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1; an isoleucine, methionine, threonine, lysine, arginine, histidine, valine, or tryptophan at position A675 of SEQ ID NO: 1; and a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1.

[0368] In some embodiments, a KOD polymerase variant comprises an isoleucine, methionine, threonine, lysine, arginine, histidine, valine, or tryptophan at position A675 of SEQ ID NO: 1; and a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1.

[0369] In some embodiments, a KOD polymerase variant comprises: an arginine at a position corresponding to A675 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1.

[0370] In some embodiments, a KOD polymerase variant comprises: an arginine at a position A675; an aspartic acid at position A747 of SEQ ID NO: 1; and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0371] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1; an arginine at a position A675; and an aspartic acid at position A747 of SEQ ID NO: 1.

[0372] In some embodiments, a KOD polymerase variant comprises an arginine at a position A675; and an aspartic acid at position A747 of SEQ ID NO: 1.

[0373] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 117. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 117. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 117. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 117. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least

[0374] #14338400v1 99.5% identity to SEQ ID NO: 117. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 117.

[0375] A347 and A747 KOD Polymerase Variants

[0376] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position A347 and a mutation corresponding to A747 of SEQ ID NO: 1.

[0377] In some embodiments, a KOD polymerase variant comprises: a threonine, asparagine, arginine, lysine, glutamine, or valine at a position corresponding to S347 of SEQ ID NO: 1; and methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at a position corresponding to A747 of SEQ ID NO: 1.

[0378] In some embodiments, a KOD polymerase variant comprises: a threonine, asparagine, arginine, lysine, glutamine, or valine at position S347 of SEQ ID NO: 1; methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1; and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0379] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1; a threonine, asparagine, arginine, lysine, glutamine, or valine at position S347 of SEQ ID NO: 1; and a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1.

[0380] In some embodiments, a KOD polymerase variant comprises a threonine, asparagine, arginine, lysine, glutamine, or valine at position S347 of SEQ ID NO: 1; and a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1.

[0381] In some embodiments, a KOD polymerase variant comprises: an arginine at a position corresponding to S347 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1.

[0382] In some embodiments, a KOD polymerase variant comprises: an arginine at position S347 and an aspartic acid at position A747 of SEQ ID NO: 1, and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0383] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1; an arginine at position S347; and an aspartic acid at position A747 of SEQ ID NO: 1.

[0384] #14338400v1 In some embodiments, a KOD polymerase variant comprises an arginine at position S347 and an aspartic acid at position A747 of SEQ ID NO: 1.

[0385] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 118. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 118. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 118. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 118. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 118. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 118.

[0386] S347, E664, and A747 KOD Polymerase Variants

[0387] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position E664, a mutation corresponding to position S347, and a mutation corresponding to A747 of SEQ ID NO: 1.

[0388] In some embodiments, a KOD polymerase variant comprises: a threonine, asparagine, arginine, lysine, glutamine, or valine at a position corresponding to S347 of SEQ ID NO: 1; a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at a position corresponding to E664 of SEQ ID NO: 1; and a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at a position corresponding to A747 of SEQ ID NO: 1.

[0389] In some embodiments, a KOD polymerase variant comprises: a threonine, asparagine, arginine, lysine, glutamine, or valine at position S347 of SEQ ID NO: 1; a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at a position E664 of SEQ ID NO: 1; and a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1; and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0390] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1; a threonine, asparagine, arginine, lysine, glutamine, or valine at position S347 of SEQ ID NO: 1; a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at position E664 of SEQ ID NO: 1; and a methionine, asparagine, leucine, histidine,

[0391] #14338400v1 aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1 of SEQ ID NO: 1.

[0392] In some embodiments, a KOD polymerase variant comprises a threonine, asparagine, arginine, lysine, glutamine, or valine at position S347 of SEQ ID NO: 1; a methionine, threonine, asparagine, lysine, serine, arginine, glutamine, valine, isoleucine, or cysteine at a position E664 of SEQ ID NO: 1; and a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at position A747 of SEQ ID NO: 1; and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0393] In some embodiments, a KOD polymerase variant comprises: an arginine at a position corresponding to S347, a lysine at a position corresponding to E664, and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1.

[0394] In some embodiments, a KOD polymerase variant comprises: an arginine at position S347, a lysine at a position E664, and an aspartic acid at position A747 of SEQ ID NO: 1.

[0395] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position S347, a lysine at a position E664, and an aspartic acid at position A747 of SEQ ID NO: 1.

[0396] In some embodiments, a KOD polymerase variant comprises an arginine at position S347, a lysine at a position E664, and an aspartic acid at position A747 of SEQ ID NO: 1.

[0397] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 119. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 119. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 119. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 119. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 119. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 119.

[0398] In some embodiments, this disclosure provides KOD polymerase variants that increase uracil tolerance of the KOD polymerase. For example, a KOD polymerase variant may increase the activity of KOD polymerase when KOD polymerase is amplifying a polynucleotide that comprises uracil (or an uracil derivative like dihydrouracil). In some embodiments, KOD

[0399] #14338400v1 polymerase mutations at positions 116, P36, P90 and V93 may increase KOD polymerase uracil tolerance.

[0400] 116 KOD Polymerase Variants

[0401] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position 116 of SEQ ID NO: 1.

[0402] In some embodiments, a KOD polymerase variant comprises: an alanine, cysteine, glutamic acid, phenylalanine, histidine, lysine, asparagine, proline, serine, valine, tryptophan, or tyrosine at a position corresponding to 116 of SEQ ID NO: 1 (e.g., I16A, I16C, I16E, I16F, I16H, I16K, I16N, I16P, I16S, I16V, I16W, or I16Y in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, a KOD polymerase variant comprises: an alanine, cysteine, glutamic acid, phenylalanine, histidine, lysine, asparagine, proline, serine, valine, tryptophan, or tyrosine at position 116 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0403] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an alanine, cysteine, glutamic acid, phenylalanine, histidine, lysine, asparagine, proline, serine, valine, tryptophan, or tyrosine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an alanine, cysteine, glutamic acid, phenylalanine, histidine, lysine, asparagine, proline, serine, valine, tryptophan, or tyrosine at position 116 of SEQ ID NO: 1.

[0404] In some embodiments, a KOD polymerase variant comprises an alanine, cysteine, glutamic acid, phenylalanine, histidine, lysine, asparagine, proline, serine, valine, tryptophan, or tyrosine at position 116 of SEQ ID NO: 1.

[0405] In some embodiments, a KOD polymerase variant comprises a alanine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a cysteine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamic acid at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant

[0406] #14338400v1 comprises a asparagine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a proline at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a serine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at a position corresponding to 116 of SEQ ID NO: 1.

[0407] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an alanine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cysteine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamic acid at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a phenylalanine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a histidine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a proline at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a serine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a valine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least

[0408] #14338400v1 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tryptophan at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tyrosine at position 116 of SEQ ID NO: 1.

[0409] In some embodiments, a KOD polymerase variant comprises a alanine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a cysteine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamic acid at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a asparagine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a proline at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a serine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at position 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at position 116 of SEQ ID NO: 1.

[0410] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 123. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 123. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 123. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 123. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 123. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 123.

[0411] In some embodiments, this disclosure provides a KOD polymerase variant comprising alanine, asparagine, cysteine, glutamic acid, histidine, lysine, phenylalanine, proline, serine, tryptophan, tyrosine, or valine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising alanine, asparagine, cysteine, glutamic acid, histidine, lysine, phenylalanine, proline, serine, tryptophan, tyrosine, or valine at position 116 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD

[0412] #14338400v1 polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine, asparagine, cysteine, glutamic acid, histidine, lysine, phenylalanine, proline, serine, tryptophan, tyrosine, or valine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine, asparagine, cysteine, glutamic acid, histidine, lysine, phenylalanine, proline, serine, tryptophan, tyrosine, or valine at position 116 of SEQ ID NO: 1.

[0413] In some embodiments, the KOD polymerase variant comprises alanine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises asparagine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises lysine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tyrosine at a position corresponding to 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises valine at a position corresponding to 116 of SEQ ID NO: 1.

[0414] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and cysteine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at

[0415] #14338400v1 least 99%) identity with SEQ ID NO: 1 and glutamic acid at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and histidine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and lysine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and phenylalanine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and proline at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and serine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tryptophan at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tyrosine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and valine at position 116 of SEQ ID NO: 1.

[0416] In some embodiments, the KOD polymerase variant comprises alanine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises asparagine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glutamic acid at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises lysine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises proline at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises tryptophan at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase

[0417] #14338400v1 variant comprises tyrosine at position 116 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises valine at position 116 of SEQ ID NO: 1.

[0418] P36 KOD Polymerase Variants

[0419] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position P36 of SEQ ID NO: 1.

[0420] In some embodiments, a KOD polymerase variant comprises: an aspartic acid, glutamic acid, phenylalanine, isoleucine, leucine, methionine, asparagine, glutamine, valine, tryptophan, or tyrosine at a position corresponding to P36 of SEQ ID NO: 1 (e.g., P36D, P36E, P36F, P36I, P36L, P36M, P36N, P36Q, P36V, P36W, or P36Y) in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, a KOD polymerase variant comprises: an aspartic acid, glutamic acid, phenylalanine, isoleucine, leucine, methionine, asparagine, glutamine, valine, tryptophan, or tyrosine at position P36 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0421] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid, glutamic acid, phenylalanine, isoleucine, leucine, methionine, asparagine, glutamine, valine, tryptophan, or tyrosine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid, glutamic acid, phenylalanine, isoleucine, leucine, methionine, asparagine, glutamine, valine, tryptophan, or tyrosine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid, glutamic acid, phenylalanine, isoleucine, leucine, methionine, asparagine, glutamine, valine, tryptophan, or tyrosine at position P36 of SEQ ID NO: 1.

[0422] In some embodiments, a KOD polymerase variant comprises an aspartic acid, glutamic acid, phenylalanine, isoleucine, leucine, methionine, asparagine, glutamine, valine, tryptophan, or tyrosine at position P36 of SEQ ID NO: 1.

[0423] In some embodiments, a KOD polymerase variant comprises an aspartic acid at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamic acid at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a

[0424] #14338400v1 histidine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a isoleucine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a leucine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a methionine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a asparagine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at a position corresponding to P36 of SEQ ID NO: 1.

[0425] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamic acid at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a phenylalanine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a histidine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an isoleucine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a leucine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at

[0426] #14338400v1 least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a valine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tryptophan at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tyrosine at position P36 of SEQ ID NO: 1.

[0427] In some embodiments, a KOD polymerase variant comprises an aspartic acid at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamic acid at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a isoleucine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a leucine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a methionine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an asparagine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a valine at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at position P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at position P36 of SEQ ID NO: 1.

[0428] #14338400v1 In some embodiments, this disclosure provides a KOD polymerase variant comprising alanine, cysteine, glycine, histidine, phenylalanine, serine, threonine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising alanine, cysteine, glycine, histidine, phenylalanine, serine, threonine at position P36 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine, cysteine, glycine, histidine, phenylalanine, serine, threonine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine, cysteine, glycine, histidine, phenylalanine, serine, threonine at position P36 of SEQ ID NO: 1.

[0429] In some embodiments, the KOD polymerase variant comprises alanine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at a position corresponding to P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at a position corresponding to P36 of SEQ ID NO: 1.

[0430] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine at position P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and cysteine at position P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glycine at position P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and histidine at position P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and

[0431] #14338400v1 phenylalanine at position P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and serine at position P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and threonine at position P36 of SEQ ID NO: 1.

[0432] In some embodiments, the KOD polymerase variant comprises alanine at position P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises cysteine at position P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises glycine at position P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises histidine at position P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises phenylalanine at position P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises serine at position P36 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises threonine at position P36 of SEQ ID NO: 1.

[0433] P90 KOD Polymerase Variants

[0434] In some embodiments, a KOD polymerase variant comprises a mutation corresponding to position P90 of SEQ ID NO: 1.

[0435] In some embodiments, a KOD polymerase variant comprises: a cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, arginine, tryptophan, or tyrosine at a position corresponding to P90 of SEQ ID NO: 1 (e.g., P90C, P90D, P90E, P90F, P90H, P90I, P90L, P90M, P90N, P90Q, P90R, P90W, P90Y in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, a KOD polymerase variant comprises: a cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, arginine, tryptophan, or tyrosine at position P90 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0436] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, arginine, tryptophan, or tyrosine at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%)

[0437] #14338400v1 identity with SEQ ID NO: 1 and a cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, arginine, tryptophan, or tyrosine at position P90 of SEQ ID NO: 1.

[0438] In some embodiments, a KOD polymerase variant comprises a cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, arginine, tryptophan, or tyrosine at position P90 of SEQ ID NO: 1.

[0439] In some embodiments, a KOD polymerase variant comprises a cysteine at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an aspartic acid at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamic acid at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an isoleucine at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a leucine at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a methionine at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an asparagine at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tyrosine at a position corresponding to P90 of SEQ ID NO: 1.

[0440] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cysteine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamic acid at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%,

[0441] #14338400v1 or at least 99%) identity with SEQ ID NO: 1 and a phenylalanine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a histidine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an isoleucine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a leucine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tryptophan at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tyrosine at position P90 of SEQ ID NO: 1.

[0442] In some embodiments, a KOD polymerase variant comprises a cysteine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a aspartic acid at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamic acid at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a phenylalanine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a histidine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a isoleucine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a leucine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a methionine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a

[0443] #14338400v1 asparagine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a glutamine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at position P90 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a tryptophan at position P90 of SEQ ID NO: 1.

[0444] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 124. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 124. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 124. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 124. In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 124. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 124.

[0445] In some embodiments, this disclosure provides a KOD polymerase variant comprising alanine, glycine, lysine, serine, threonine, or valine at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments, this disclosure provides a KOD polymerase variant comprising alanine, glycine, lysine, serine, threonine, valine at position P90 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine, glycine, lysine, serine, threonine, valine at a position corresponding to P90 of SEQ ID NO: 1. In some embodiments,...

Claims

1. CLAIMSWhat is claimed is:

1. A Thermococcus kodakarensis (KOD) polymerase variant, comprising:(i) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to E325 of SEQ ID NO: 1(ii) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, leucine, lysine, phenylalanine, proline, threonine, tryptophan, tyrosine, or valine at a position corresponding to S347 of SEQ ID NO: 1(iii) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to F356 of SEQ ID NO: 1(iv) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to L396 of SEQ ID NO: 1(v) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to 1488 of SEQ ID NO: 1(vi) an arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, methionine, proline, serine, tryptophan, or tyrosine at a position corresponding to F587 of SEQ ID NO: 1(vii) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, or tyrosine at a position corresponding to V589 of SEQ ID NO: 1(viii) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, or valine at a position corresponding to T605 of SEQ ID NO: 1(ix) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to G634 of SEQ ID NO: 1(x) an alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to R641 of SEQ ID NO: 1#14338400v1(xi) an alanine, arginine, asparagine, aspartic acid, glycine, histidine, isoleucine, leucine, methionine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to E664 of SEQ ID NO: 1(xii) an arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to A675 of SEQ ID NO: 1(xiii) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to G677 of SEQ ID NO: 1(xiv) an arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to A747 of SEQ ID NO: 1(xv) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to F748 of SEQ ID NO: 1.

2. The KOD polymerase variant of claim 1, comprising:(i) a methionine, threonine, lysine, serine, arginine, leucine, proline, histidine, glutamine, valine, alanine, glycine, phenylalanine, cysteine, or tyrosine at a position corresponding to E325 of SEQ ID NO: 1;(ii) a threonine, asparagine, arginine, lysine, glutamine, or valine at a position corresponding to S347 of SEQ ID NO: 1;(iii) an isoleucine, methionine, asparagine, lysine, serine, arginine, leucine, valine, alanine, aspartic acid, glycine, or cysteine at a position corresponding to F356 of SEQ ID NO: 1;(iv) a threonine, lysine, serine, arginine, proline, histidine, glutamine, alanine, aspartic acid, glutamic acid, glycine, tyrosine, or cysteine at a position corresponding to L396 of SEQ ID NO: 1;(v) an arginine, asparagine, lysine, glutamine, alanine, aspartic acid, or glycine at a position corresponding to 1488 of SEQ ID NO: 1;(vi) a methionine, asparagine, lysine, histidine, glutamine, tyrosine, cysteine, or glycine at a position corresponding to F587 of SEQ ID NO: 1;(vii) an asparagine, arginine, alanine, glycine, phenylalanine, tyrosine, or tryptophan at a position corresponding to V589 of SEQ ID NO: 1;#14338400v1(viii) an isoleucine, proline, histidine, valine, alanine, phenylalanine, tyrosine, cysteine, or tryptophan at a position corresponding to T605 of SEQ ID NO: 1;(ix) an asparagine, lysine, arginine, leucine, proline, glutamine, aspartic acid, glutamic acid, phenylalanine, tyrosine, cysteine, tryptophan, isoleucine, threonine, serine, histidine, valine, or alanine at a position corresponding to G634 OF SEQ ID NO: 1;(x) a proline, glutamic acid, or alanine at a position corresponding to R641 of SEQ ID NO: 1;(xi) a methionine, threonine, asparagine, serine, arginine, valine, or isoleucine at a position corresponding to E664 of SEQ ID NO: 1;(xii) an isoleucine, threonine, lysine, arginine, histidine, valine, or tryptophan at a position corresponding to A675 of SEQ ID NO: 1;(xiii) a methionine, threonine, serine, arginine, leucine, proline, histidine, glutamine, phenylalanine, lysine, or alanine at a position corresponding to G677 of SEQ ID NO: 1;(xiv) a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at a position corresponding to A747 of SEQ ID NO: 1; and / or(xv) a lysine, arginine, histidine, aspartic acid, tyrosine, tryptophan, or serine at a position corresponding to F748 of SEQ ID NO: 1.

3. The KOD polymerase variant of claim 2, comprising a methionine, threonine, lysine, serine, arginine, leucine, proline, histidine, glutamine, valine, alanine, glycine, phenylalanine, cysteine, or tyrosine at a position corresponding to E325 of SEQ ID NO: 1.

4. The KOD polymerase variant of claim 3, comprising a valine at a position corresponding to E325 of SEQ ID NO: 1.

5. The KOD polymerase variant of any one of claims 2-4, comprising a threonine, asparagine, arginine, lysine, glutamine, or valine at a position corresponding to S347 of SEQ IDNO: 1.

6. The KOD polymerase variant of claim 5, comprising an arginine at a position corresponding to S347 of SEQ ID NO: 1.#14338400v17. The KOD polymerase variant of any one of claims 2-6, comprising an isoleucine, methionine, asparagine, lysine, serine, arginine, leucine, valine, alanine, aspartic acid, glutamine, or cysteine at a position corresponding to F356 of SEQ ID NO: 1.

8. The KOD polymerase variant of claim 7, comprising an asparagine at a position corresponding to F356 of SEQ ID NO: 1.

9. The KOD polymerase variant of any one of claims 2-8, comprising a threonine, lysine, serine, arginine, proline, histidine, glutamine, alanine, aspartic acid, glutamic acid, glycine, tyrosine, or cysteine at a position corresponding to L396 of SEQ ID NO: 1.

10. The KOD polymerase variant of claim 9, comprising a glutamic acid at a position corresponding to L396 of SEQ ID NO: 1.

11. The KOD polymerase variant of claim any one of claims 2-10, comprising an arginine, asparagine, lysine, glutamine, alanine, aspartic acid, or glycine at a position corresponding to 1488 of SEQ ID NO: 1.

12. The KOD polymerase variant of claim 11, comprising an arginine at a position corresponding to 1488 of SEQ ID NO: 1.

13. The KOD polymerase variant of any one of claims 2-12, comprising a methionine, asparagine, lysine, histidine, glutamine, tyrosine, cysteine, or glycine at a position corresponding to F587 of SEQ ID NO: 1.

14. The KOD polymerase variant of claim 13, comprising a methionine at a position corresponding to F587 of SEQ ID NO: 1.

15. The KOD polymerase variant of claim any one of claims 2-14, comprising an asparagine, arginine, alanine, glycine, phenylalanine, tyrosine, or tryptophan at a position corresponding to V589 of SEQ ID NO: 1.

16. The KOD polymerase variant of claim 15, comprising a tryptophan at a position corresponding to V589 of SEQ ID NO: 1.#14338400v117. The KOD polymerase variant of any one of claims 2-16, comprising an isoleucine, proline, histidine, valine, alanine, phenylalanine, tyrosine, cysteine, or tryptophan at a position corresponding to T605 of SEQ ID NO: 1.

18. The KOD polymerase variant of claim 17, comprising a tyrosine at a position corresponding to T605 of SEQ ID NO: 1.

19. The KOD polymerase variant of any one of claims 2-18, comprising an asparagine, lysine, arginine, leucine, proline, glutamine, aspartic acid, glutamic acid, phenylalanine, tyrosine, cysteine, tryptophan, isoleucine, threonine, serine, histidine, valine, or alanine at a position corresponding to G634 OF SEQ ID NO: 1.

20. The KOD polymerase variant of claim 19, comprising an aspartic acid at a position corresponding to G634 OF SEQ ID NO: 1.

21. The KOD polymerase variant of any one of claims 2-20, comprising a proline, glutamic acid, or alanine at a position corresponding to R641 of SEQ ID NO: 1.

22. The KOD polymerase variant of claim 21, comprising a proline at a position corresponding to R641 of SEQ ID NO: 1.

23. The KOD polymerase variant of any one of claims 2-22, comprising a methionine, threonine, asparagine, serine, arginine, valine, or isoleucine at a position corresponding to E664 of SEQ ID NO: 1.

24. The KOD polymerase variant of claim 23, comprising a lysine at a position corresponding to E664 of SEQ ID NO: 1.

25. The KOD polymerase variant of any one of claims 2-24, comprising an isoleucine, threonine, lysine, arginine, histidine, valine, or tryptophan at a position corresponding to A675 of SEQ ID NO: 1.

26. The KOD polymerase variant of claim 25, comprising an arginine at a position corresponding to A675 of SEQ ID NO: 1.#14338400v127. The KOD polymerase variant of any one of claims 2-26, comprising a methionine, threonine, serine, arginine, leucine, phenylalanine, histidine, glutamine, proline, lysine, or alanine at a position corresponding to G677 of SEQ ID NO: 1.

28. The KOD polymerase variant of claim 27, comprising a proline at a position corresponding to G677 of SEQ ID NO: 1.

29. The KOD polymerase variant of any one of claims 2-28, comprising a methionine, asparagine, leucine, histidine, aspartic acid, phenylalanine, tyrosine, tryptophan, isoleucine, or glutamic acid at a position corresponding to A747 of SEQ ID NO: 1.

30. The KOD polymerase variant of claim 29, comprising an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1.

31. The KOD polymerase variant of any one of claims 2-30, comprising a lysine, arginine, histidine, aspartic acid, tyrosine, tryptophan, or serine at a position corresponding to F748 of SEQ ID NO: 1.

32. The KOD polymerase variant of claim 31, comprising a tyrosine at a position corresponding to F748 of SEQ ID NO: 1.

33. The KOD polymerase variant of claim 1 or 2, further comprising one or more of: an isoleucine, leucine or valine at a position corresponding to F587 of SEQ ID NO: 1; a histidine or glutamine at a position corresponding to V589 of SEQ ID NO: 1; a lysine, glutamine, or cysteine at a position corresponding to E664 of SEQ ID NO: 1; or a methionine at a position corresponding to A675 of SEQ ID NO: 1.

34. The KOD polymerase variant of claim 1, comprising an amino acid sequence of any one of SEQ ID NOs: 2-6.

35. The KOD polymerase variant of claim 1, comprising an amino acid sequence of any one of SEQ ID NOs: 7-9.#14338400v136. The KOD polymerase variant of claim , comprising an amino acid sequence of SEQ ID NOs: 10.

37. The KOD polymerase variant of claim , comprising an amino acid sequence of any one of SEQ ID NOs: 11-23.

38. The KOD polymerase variant of claim , comprising an amino acid sequence of SEQ ID NO: 24.

39. The KOD polymerase variant of claim , comprising an amino acid sequence of any one of SEQ ID NOs: 25-34.

40. The KOD polymerase variant of claim , comprising an amino acid sequence of any one of SEQ ID NOs: 35-43.

41. The KOD polymerase variant of claim , comprising an amino acid sequence of any one of SEQ ID NOs: 44-52.

42. The KOD polymerase variant of claim , comprising an amino acid sequence of any one of SEQ ID NOs: 53-70.

43. The KOD polymerase variant of claim , comprising an amino acid sequence of any one of SEQ ID NOs: 71-72.

44. The KOD polymerase variant of claim , comprising an amino acid sequence of any one of SEQ ID NOs: 73-80.

45. The KOD polymerase variant of claim , comprising an amino acid sequence of any one of SEQ ID NOs: 81-88.

46. The KOD polymerase variant of claim , comprising an amino acid sequence of any one of SEQ ID NOs: 89-97.#14338400v147. The KOD polymerase variant of claim 1, comprising an amino acid sequence of any one of SEQ ID NOs: 98-105.

48. The KOD polymerase variant of claim 1, comprising an amino acid sequence of any one of SEQ ID NOs: 106-111.

49. The KOD polymerase variant of claim 2, comprising:(i) a lysine at a position corresponding to E664 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1;(ii) a tyrosine at a position corresponding to T605 and a lysine at a position corresponding to E664 of SEQ ID NO: 1;(iii) a methionine at a position corresponding to F587 and a lysine at a position corresponding to E664 of SEQ ID NO: 1;(iv) an arginine at a position corresponding to S347 and a lysine at a position corresponding to E664 of SEQ ID NO: 1;(v) a proline at a position corresponding to G677 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1;(vi) an arginine at a position corresponding to A675 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1;(vii) an arginine at a position corresponding to S347 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1; or(viii) an arginine at a position corresponding to S347, a lysine at a position corresponding to E664, and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1.

50. The KOD polymerase variant of claim 49, comprising a lysine at a position corresponding to E664 and an aspartic acid at a position corresponding to A747 of SEQ ID NO:1.

51. The KOD polymerase variant of claim 50, comprising an amino acid sequence of SEQ ID NO: 112.

52. The KOD polymerase variant of claim 49, comprising a tyrosine at a position corresponding to T605 and a lysine at a position corresponding to E664 of SEQ ID NO: 1.#14338400v153. The KOD polymerase variant of claim 52, comprising an amino acid sequence of SEQ ID NO: 113.

54. The KOD polymerase variant of claim 49, comprising a methionine at a position corresponding to F587 and a lysine at a position corresponding to E664 of SEQ ID NO: 1.

55. The KOD polymerase variant of claim 54, comprising an amino acid sequence of SEQ ID NO: 114.

56. The KOD polymerase variant of claim 49, comprising an arginine at a position corresponding to S347 and a lysine at a position corresponding to E664 of SEQ ID NO: 1.

57. The KOD polymerase variant of claim 56, comprising an amino acid sequence of SEQ ID NO: 115.

58. The KOD polymerase variant of claim 49, comprising a proline at a position corresponding to G677 and an aspartic acid at a position corresponding to A747 of SEQ ID NO:1.

59. The KOD polymerase variant of claim 58, comprising an amino acid sequence of SEQ ID NO: 116.

60. The KOD polymerase variant of claim 49, comprising an arginine at a position corresponding to A675 and an aspartic acid at a position corresponding to A747 of SEQ ID NO:1.

61. The KOD polymerase variant of claim 60, comprising an amino acid sequence of SEQ ID NO: 117.

62. The KOD polymerase variant of claim 49, comprising an arginine at a position corresponding to S347 and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1.#14338400v163. The KOD polymerase variant of claim 62, comprising an amino acid sequence of SEQ ID NO: 118.

64. The KOD polymerase variant of claim 49, comprising an arginine at a position corresponding to S347, a lysine at a position corresponding to E664, and an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1.

65. The KOD polymerase variant of claim 64, comprising an amino acid sequence of SEQ ID NO: 119.

66. The KOD polymerase variant of any one of claims 1-65, comprising a mutation at a position corresponding to one or more of 116, P36, P90, or V93 of SEQ ID NO: 1.

67. The KOD polymerase variant of claim 66, comprising:(i) an alanine, cysteine, glutamic acid, phenylalanine, histidine, lysine, asparagine, proline, serine, valine, tryptophan, or tyrosine at a position corresponding to 116 of SEQ ID NO: 1;(ii) an aspartic acid, glutamic acid, phenylalanine, isoleucine, leucine, methionine, asparagine, glutamine, valine, tryptophan, tyrosine at a position corresponding to P36 of SEQ ID NO: 1;(iii) a cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, arginine, tryptophan, or tyrosine at a position corresponding to P90 of SEQ ID NO: 1; and / or(iv) alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, tryptophan, or tyrosine at a position corresponding to V93 of SEQ ID NO: 1.

68. A KOD polymerase variant, comprising:(i) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to 116 of SEQ ID NO: 1;(ii) an alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to P36 of SEQ ID NO: 1;#14338400v1(iii) an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine at a position corresponding to P90 of SEQ ID NO: 1; and / or(iv) an alanine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, or tyrosine at a position corresponding to V93 of SEQ ID NO: 1.

69. The KOD polymerase variant of claim 68, comprising:(i) an alanine, cysteine, glutamic acid, phenylalanine, histidine, lysine, asparagine, proline, serine, valine, tryptophan, or tyrosine at a position corresponding to 116 of SEQ ID NO: 1;(ii) an aspartic acid, glutamic acid, phenylalanine, isoleucine, leucine, methionine, asparagine, glutamine, valine, tryptophan, tyrosine at a position corresponding to P36 of SEQ ID NO: 1;(iii) a cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, arginine, tryptophan, or tyrosine at a position corresponding to P90 of SEQ ID NO: 1; and / or(iv) alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, leucine, methionine, asparagine, proline, serine, threonine, tryptophan, or tyrosine at a position corresponding to V93 of SEQ ID NO: 1.

70. The KOD polymerase variant of claim 69, comprising an alanine, cysteine, glutamic acid, phenylalanine, histidine, lysine, asparagine, proline, serine, valine, tryptophan, or tyrosine at a position corresponding to 116 of SEQ ID NO: 1.

71. The KOD polymerase variant of claim 69 or claim 70, comprising a tryptophan at a position corresponding to 116 of SEQ ID NO: 1.

72. The KOD polymerase variant of any one of claims 69-71, comprising an aspartic acid, glutamic acid, phenylalanine, isoleucine, leucine, methionine, asparagine, glutamine, valine, tryptophan, tyrosine at a position corresponding to P36 of SEQ ID NO: 1.

73. The KOD polymerase variant of any one of claims 69-72, comprising a cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine,#14338400v1glutamine, arginine, tryptophan, or tyrosine at a position corresponding to P90 of SEQ ID NO: 1.

74. The KOD polymerase variant of any one of claims 69-73, comprising a tryptophan at a position corresponding to P90 of SEQ ID NO: 1.

75. The KOD polymerase variant of any one of claims 69-74, comprising alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, leucine, methionine, asparagine, proline, serine, threonine, tryptophan, or tyrosine at a position corresponding to V93 of SEQ ID NO: 1.

76. The KOD polymerase variants of any one of claims 69-75 further comprising:(i) a histidine, lysine or arginine at a position corresponding to P36 of SEQ ID NO: 1; and(ii) a histidine, lysine, glutamine, or arginine at a position corresponding to V93 of SEQ ID NO: 1.

77. A KOD polymerase variant, comprising:(i) an arginine at a position corresponding to S347 of SEQ ID NO: 1; or(ii) an aspartic acid at a position corresponding to A747 of SEQ ID NO: 1.

78. The KOD polymerase variant of claim 77, comprising the arginine at the position corresponding to S347 of SEQ ID NO: 1.

79. The KOD polymerase variant of claim 78, comprising the aspartic acid at the position corresponding to A747 of SEQ ID NO: 1.

80. The KOD polymerase variant of any one of claims 77-79, comprising a lysine at the position corresponding to E664 of SEQ ID NO: 1.

81. The KOD polymerase variant of claim 77, comprising the aspartic acid at the position corresponding to A747 of SEQ ID NO: 1.

82. The KOD polymerase variant of claim 81, comprising a lysine at position corresponding to E664 of SEQ ID NO: 1.#14338400v183. The KOD polymerase variant of claim 77 comprising:(i) the arginine at the position corresponding to S347 of SEQ ID NO: 1;(ii) the aspartic acid at the position corresponding to A747 of SEQ ID NO: 1; and(ii) a lysine at a position corresponding to E664 of SEQ ID NO: 1.

84. The KOD polymerase variant of any one of claims 77-83, further comprising:(a) a glutamine at a position corresponding to V93 of SEQ ID NO: 1;(b) a tryptophan at a position corresponding to P90 of SEQ ID NO: 1; or(c) a tryptophan at a position corresponding to 116 of SEQ ID NO: 1.

85. The KOD polymerase variant of claim 84, comprising the glutamine at the position corresponding to V93 of SEQ ID NO: 1.

86. The KOD polymerase variant of claim 84 or claim 85, comprising the tryptophan at the position corresponding to P90 of SEQ ID NO: 1.

87. The KOD polymerase variant of any one of claims 84-86, comprising the tryptophan at the position corresponding to 116 of SEQ ID NO: 1.

88. The KOD polymerase variant of claim 77, comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 9, 102, 112, or 115-119.

89. The KOD polymerase variant of claim 77, comprising an amino acid sequence of any one of SEQ ID NOs: 9, 102, 112, or 115-119.

90. The KOD polymerase variant of claim 77, comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 120-122.

91. The KOD polymerase variant of claim 77, comprising an amino acid sequence of any one of SEQ ID NOs: 120-122.

92. The KOD polymerase variant of claim 77, comprising an amino acid sequence of SEQ ID NO: 120.#14338400v193. The KOD polymerase variant of claim 77, comprising an amino acid sequence of SEQ ID NO: 121.

94. The KOD polymerase variant of claim 77, comprising an amino acid sequence of SEQ ID NO: 122.

95. The KOD polymerase variant of claim 77, comprising:(i) the tryptophan at the position corresponding to P90 of SEQ ID NO: 1;(ii) the tryptophan at the position corresponding to 116 of SEQ ID NO: 1; and(iii) a glutamine at a position corresponding to V93 of SEQ ID NO: 1.

96. The KOD polymerase variant of claim 77, comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 120-125.

97. The KOD polymerase variant of claim 77, comprising an amino acid sequence of any one of SEQ ID NOs: 120-125.

98. The KOD polymerase variant of claim 1, comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2-125.

99. A polynucleotide variant comprising a nucleic acid encoding the KOD polymerase variant of any one of claims 1-98.

100. A method for nucleic acid synthesis, the method comprising contacting a nucleic acid template with primers, deoxynucleotide triphosphates (dNTPs), and the KOD polymerase variant of any one of claims 1-98.

101. The method of claim 100, wherein the nucleic acid synthesis is performed in more than 150 mM of potassium ion.

102. The method of claim 100, wherein the nucleic acid synthesis is performed in at least 300 mM of potassium ion.#14338400v1103. The method of claim 100, wherein the nucleic acid synthesis is performed in less than1.5 mM of magnesium ion.#14338400v1

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