Reverse transcriptase mutants and uses thereof

Abstract

The present disclosure provides Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) variants. The present disclosure further provides amino acid positions for mutagenesis of MMLV RTase as well as nucleic acids, kits, compositions, fusion proteins, and methods including MMLV RTase variants.

Description

[0001] 2024-24213-P-WO-P15916WO01 REVERSE TRANSCRIPTASE MUTANTS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATION(S) This application claims the benefit of U. S. Provisional Application No.

[0002] 63 / 739,262 filed December 27, 2024 for “REVERSE TRANSCRIPTASE MUTANTS AND USES THEREOF” by S. F. Beaudoin and C. A. Vakulskas.

[0003] FIELD OF THE INVENTION

[0004] The present disclosure relates to reverse transcriptase enzymes and, more particularly, to mutant variants of Moloney murine leukemia virus reverse transcriptase and uses thereof.

[0005] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0006] The present application includes a Sequence Listing which has been submitted electronically as an XML document in the ST.26 format via Patent Center and concurrent with the filing of the application, and which is hereby incorporated by reference in its entirety. The XML document is named "2024-24213-P-US-D0487-P15916US01.xml,” was created on December 11, 2024, and is 845,224 bytes in size.

[0007] BACKGROUND

[0008] Reverses transcriptase (RTase) enzymes facilitate reverse transcription of ribonucleic acid (RNA) into copy deoxyribonucleic acid (cDNA). RTases are encoded and used by retroviruses to reverse-transcribe RNA genomic material into cDNA, which can then be integrated into the host genome. RTase enzymes are often used in laboratory applications to produce cDNA for a variety of downstream applications, including quantitative PCR, gene expression analysis, isolated RNA sequencing, gene cloning, and cDNA library creation.

[0009] SUMMARY

[0010] An example of an isolated Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutant according to the present disclosure includes an isolated MMLV RTase comprising an amino acid sequence as set forth in any of SEQ ID NOs: 278-432.

[0011] An MMLV RTase variant comprising a substitution at a position corresponding to least one of position 9, position 157, position 159, position 173, position 287, position 333, position 433, position 435, position 467, position 490, position 502, position 629, or position 646 of any of SEQ ID NOs: 275-277.An example of a nucleotide according to the present disclosure includes a nucleotide encoding an MMLV RTase disclosed herein.

[0012] An example of a cDNA according to the present disclosure includes a cDNA encoding an MMLV RTase disclosed herein.

[0013] An example of a kit according to the present disclosure includes a kit comprising an MMLV RTase disclosed herein.

[0014] An example of a composition according to the present disclosure includes a composition comprising an MMLV RTase disclosed herein.

[0015] An isolated prime editor fusion protein according to the present disclosure includes a prime editor fusion protein comprising a nuclease domain and a reverse transcriptase domain having a sequence of an MMLV RTase disclosed herein.

[0016] An example of a method of synthesizing cDNA according to the present disclosure includes providing a ribonucleic acid template and contacting the ribonucleic acid template with an MMLV RTase disclosed herein to synthesize cDNA.

[0017] An example of a method of gene editing according to the present disclosure includes providing a sample including a double- stranded target DNA sequence and contacting the double- stranded target DNA sequence with a prime editor. The prime editor includes first domain comprising a nucleic acid programmable DNA binding protein, a second domain comprising the MMLV RTase disclosed herein, and a prime editing guide RNA.

[0018] An example of a method of performing reverse transcriptase-polymerase chain reaction (RT-PCR) according to the present disclosure includes providing a ribonucleic acid template, contacting the ribonucleic acid template with an MMLV RTase of the present disclosure to synthesize cDNA, and contacting the cDNA with a DNA polymerase to amplify the cDNA.

[0019] The present summary is provided only by way of example, and not limitation. Other aspects of the present disclosure will be appreciated in view of the entirety of the present disclosure, including the entire text, claims, and accompanying figures.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow diagram of an example of a method of synthesizing cDNA. While the above-identified figures set forth one or more examples of the present disclosure, other examples are also contemplated, as noted in the discussion. In all cases, this disclosure presents the invention by way of representation and not limitation. It should be understood that numerous other modifications and examples can be devised bythose skilled in the art, which fall within the scope and spirit of the principles of the invention. The figures may not be drawn to scale, and applications and examples of the present invention may include features and components not specifically shown in the drawings.

[0021] DETAILED DESCRIPTION

[0022] The present disclosure relates to Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) enzymes. In particular, the present disclosure provides mutant variants of MMLV RTase and sequences encoding mutant variants of MMLV RTase. The present disclosure also provides various mutagenesis primers that can be used to perform site-directed mutagenesis of an MMLV RTase nucleotide sequence to create a sequence encoding an MMLV RTase variant disclosed herein. The disclosure further provides various methods and kits that use the MMLV RTase variants disclosed herein to synthesize copy deoxyribonucleic acid (cDNA) from ribonucleic acid (RNA) template sequences.

[0023] Various RTase enzymes are used in research applications to synthesize cDNA for, e.g., polymerase chain reaction (PCR), gene-editing techniques, cDNA library construction, RNA sequencing, etc. MMLV RTase is characterized by low RNase H activity and a higher fidelity as compared to other common RTases used in laboratory applications, such as Avian Myeloblastosis Virus (AMV) RTase. The reduced RNase H activity reduces the rate at which MMLV RTase degrades RNA templates and allows MMLV RTases to be used for the reverse transcription of long RNAs (>5 kilobase) and in prime editing applications. Various MMLV RTases with further reduced RNase H activity have been constructed to provide additional reductions to RNA template degradation and, consequently, additional improvements to cDNA synthesis.

[0024] However, cDNA synthesis by MMLV RTase can be impeded by the presence of secondary structures in RNA templates and, notably, wildtype MMLV RTase has significantly reduced activity at the elevated temperatures useful for disrupting RNA. In particular, the optimal temperature for wildtype MMLV RTase activity is 37 °C. However, reaction temperatures of over 45 °C or, in some examples, above 50 °C are recommended to disrupt RNA secondary structures and, further, to reduce non-specific priming (i.e., by reducing mismatch duplex stability). To improve the thermostability of MMLV RTase, various methods further attempt to substitute Mn2+for Mg2+as the RTase cofactor by altering reaction conditions (e.g., by performing cDNA synthesis in high Mn2+conditions and in the absence of Mg2+) and / or by using variant MMLV RTases with enhanced affinity for Mn2+, but these methods generally result in increased error rates.As will be explained in more detail subsequently, and particularly with respect to the data presented in the discussion of Examples 3-5, the MMLV RTase mutant variants disclosed herein have improved activity and thermostability as compared to wildtype MMLV RTase. Notably, the mutant variants of MMLV RTase also have improved activity and thermostability as compared to other existing mutant variants of MMLV RTase. The enhanced activity and thermostability of the MMLV RTase mutants disclosed herein is advantageous in various applications, including in PCR and prime editing applications.

[0025] One of skill in the art will recognize that cDNA molecules made using the MMLV RTase mutants of the disclosure can be used in a variety of additional downstream applications. For example, amplification methods may include one-step PCR, two-step PCR, real-time or quantitative PCR, hot-start PCR, nested PCR, touch down PCR, differential display PCR (DDRT-PCR), microarray technologies, inverse PCR, Rapid amplification of PCR ends (RACE or anchored PCR), multiplex PCR, and site directed PCR mutagenesis. Synthesized cDNA and cDNA libraries created with the MMLV RTase mutants of the disclosure can be used in cloning and / or sequencing for further characterization. One of skill in the art will recognize that nucleic acid amplification using cDNA prepared with the MMLV RTase mutants of the disclosure may include additional techniques not listed herein.

[0026] In some examples, an MMLV RTase variant according to the present disclosure can be an isolated MMLV RTase variant having a sequence of any of the MMLV RTase variants disclosed herein or a sequence that is substantially identical to any of the MMLV RTase variants disclosed herein. In yet further examples, an MMLV RTase variant according to the present disclosure can be an isolated MMLV RTase having one or more useful mutations disclosed herein.

[0027] An MMLV RTase mutant according to the present disclosure can be included in a kit for use in reverse transcription or related technologies. These kits include one or more of the following: an MMLV RTase mutant enzyme, reagents and buffers for conducting a reverse transcriptase reaction, a box, vial tubes, ampules, and the like. Kits can also include instructions for use of the kit for practicing any of the methods disclosed herein or other methods known to those of skill in the art.

[0028] Further, an MMLV RTase mutant according to the present disclosure can be a domain of a prime editor fusion protein for use in a clustered regularly interspaced short palindromic repeat (CRISPR) technique or another suitable gene-editing technique.More particularly, a fusion protein of an MMLV RTase mutant according to the present disclosure and a CRISPR-associated protein (Cas), such as a Cas9 protein (e.g., Cas9 nickase), can be constructed using any suitable technique (e.g., a suitable cloning technique). The MMLV RTase domain of the resultant fusion protein can be used to perform reverse transcription (i.e., cDNA synthesis) as part of a prime editing technique. In these examples, the MMLV RTase mutant can be included in a kit for prime editing and, optionally, can be provided with one or more prime editing guide RNAs (pegRNAs).

[0029] FIG. 1 is a flow diagram of method 100, which is an example of a method of synthesizing cDNA. Method 100 includes steps of 102-104 of providing a ribonucleic acid template (step 102) and contacting the ribonucleic acid template with an MMLV RTase variant (104). Method 100 can be performed as part of a PCR method, a prime editing method, or any other suitable method using a reverse transcriptase to produce a cDNA.

[0030] In step 102, a ribonucleic acid template is provided. The template can be provided by any suitable method and, in some examples, can be added to a reaction mixture including one or more buffer components, deoxynucleotide triphosphates (dNTPs), and / or any other suitable components.

[0031] In step 104, the ribonucleic acid template provided in step 120 is contacted with an MMLV RTase variant. Contacting ribonucleic acid template with the MMLV RTase variant allows the MMLV RTase variant to reverse transcribe the ribonucleic acid template to produce a cDNA.

[0032] Where method 100 is used to perform a PCR technique, the template provided in step 102 can be provided, for example, as part of a reaction mixture including one or more buffer components, deoxynucleotide triphosphates (dNTPs), etc. In step 104, the MMLV RTase variant can be added to the reaction mixture, thereby allowing the MMLV RTase variant to contact the template and synthesize cDNA. The cDNA can subsequently be amplified in a separate reaction (i.e., as part of a two-step procedure) or in the same reaction (i.e., as part of a one-step procedure) using a DNA polymerase. Amplified cDNA product can, in some use cases, be quantified, analyzed, etc. using any suitable technique (e.g., a qPCR technique).

[0033] Where method 100 is used to perform a prime editing technique, the ribonucleic acid template provided in step 102 can be a template included in a pegRNA. The pegRNA can include a protospacer element for targeting a particular genomic region for gene-editing. In step 104, nuclease domain of the prime editor fusion protein (e.g., aCas9 nickase domain) can introduce a single-strand cut to allow a primer-binding sequence (PBS) to anneal to an end of the nicked strand. The PBS can be adjacent or substantially to the RNA template in the pegRNA and is complementary to the free 3’ end of the nicked strand, allowing the free 3’ end to be used as a primer for cDNA synthesis. After the PBS anneals to the end of the nicked strand, the variant MMLV RTase domain of the prime editor fusion protein then contacts the RNA template to perform cDNA synthesis. The resultant heteroduplex can then be resolved via a mismatch repair mechanism.

[0034] EXAMPLES

[0035] The following examples are illustrative and are not intended to limit the scope of the invention.

[0036] Example 1 — Preparation of MMLV RTase mutants by Site Directed Mutagenesis

[0037] The MMLV RTase variants described herein were prepared by introducing various mutations to a base construct that encodes a variant of MMLV RTase having thirteen mutations (SEQ ID NO: 119, encoding SEQ ID NO: 277) relative to the wildtype sequence (SEQ ID NO: 117, encoding SEQ ID NO: 275). The base construct having the amino acid sequence set forth in SEQ ID NO: 277 (and encoded by the DNA sequence set forth in SEQ ID NO: 119) is referred to herein as the “M63 variant” and the “M63 MMLV RTase.” The M63 variant is a variant of wild-type MMLV RTase (SEQ ID NO: 117, encoding SEQ ID NO: 275) including three mutations contained within SuperScript II (SSII) RTase (Invitrogen; SEQ ID NO: 118, encoding SEQ ID NO: 276) and ten additional mutations identified in U. S. Patent Publication No. 2023 / 0031558. The three mutations of the SuperScript II RTase (SEQ ID NO: 276) have been shown to reduce RNase activity. Further, the additional ten mutations of the M63 variant (SEQ ID NO: 275) have been shown to improve activity and thermostability as compared to wild-type MMLV RTase (U. S. Pat. Pub. No. 2023 / 0031558).

[0038] The mutations contained by the variants disclosed herein (SEQ IDs: 278-432) were obtained by site-directed mutagenesis of the M63 variant using standard PCR conditions and relevant primers. Primers used for construction of each mutation are provided in Table 1. The resultant plasmids were transformed into E. coli BL21(DE3) for expression. The M63 variant base construct was contained in a pET28a vector and all variant amino acid sequences contain an N-terminal 20mer 6x His-tag having a sequence of “MGSSHHHHHHSSGLVPRGSH.” However, for clarity and convenience, mutationpositions herein are referred to by their position relative to the methionine following the N-terminal 20mer encoded by the pET28a vector.

[0039] The oligonucleotide primers in Table 1 include “TOP” or “BTM” designations, which refer to top and bottom primers used for site-directed mutagenesis. Table 1 includes 58 pairs of oligonucleotide primers for creating 58 mutations to the MMLV RTase M63 variant. Each pair has the same “Primer Name,” except for the “TOP” or “BTM” designations, and SEQ IDs 1-116 are ordered such that SEQ IDs 1-58 are paired to respective ones of SEQ IDs 59-116. One pair of corresponding primers (e.g., SEQ ID 1 and SEQ ID 59, SEQ ID 2 and SEQ ID 60, etc.) were used to introduce each mutation via site-directed mutagenesis. In the examples in Table 1, each pair of corresponding primers are reverse complements. Although all MMLV variants disclosed herein were obtained by site-directed mutagenesis of the M63 MMLV RTase sequence (SEQ ID NO: 119), for clarity and convenience, mutations in MMLV RTase sequences are identified using amino acid identities of the wildtype sequence.

[0040] TABLE 1

[0041] Sequences of oligonucleotide primers used for site-directed mutagenesis of the M63 variant of MMLV RTase. SEQ ID Primer Name Primer Sequence (5' - 3')

[0042] 1 MMLV_M63 GCAGCCATATGACTTTAAATATTGAGGATCCCCATCGTTTACATGAGACATCAAAAGAAC E7P TOP

[0043] 2 MMLV_M63 CCATATGACTTTAAATATTGAGGATGAGCATTTATTACATGAGACATCAAAAGAACCCGAC R9L TOP

[0044] 3 MMLV_M63 CCATATGACTTTAAATATTGAGGATGAGCATGCGTTACATGAGACATCAAAAGAACCCGAC R9A TOP

[0045] 4 MMLV_M63 GCGTAGAAGACATCCATCCGACTTATCCTAATCCTTATAATCTGTTATCAGGCCT V129Y TOP

[0046] 5 MMLV_M63 CCGACTGTACCTAATCCTTATAATCTGGCGTCAGGCCTGCCCCCATCGC

[0047] L136ATOP

[0048] 6 MMLV_M63 CGACTGTACCTAATCCTTATAATCTGTTATTCGGCCTGCCCCCATCGC

[0049] S137F TOP

[0050] 7 MMLV_M63 GACTGTACCTAATCCTTATAATCTGTTATCATCTCTGCCCCCATCGCACCAAT

[0051] G138S TOP

[0052] 8 MMLV_M63 CAGTATTAGACTTGAAAGACGCGTTCGTATGCCTGCGTCTGCACCCAA

[0053] F156V TOP

[0054] 9 MMLV_M63 GTATTAGACTTGAAAGACGCGTTCTTTGCGCTGCGTCTGCACCCAACGT

[0055] C157A TOP

[0056] 10 MMLV_M63 CCGCTGTTTGCGTTCGAATGGGAGGATCCTGAAATGGGAATTTCGGGT

[0057] R173E TOP

[0058] 11 MMLV_M63 GCCCCAGGGCTTTAAAAACAGCGCGACATTGTTCGATGAAGCACTTCACCGT

[0059] P196A TOPMMLV_M63 GCCCCAGGGCTTTAAAAACAGCTTAACATTGTTCGATGAAGCACTTCACCGT

[0060] P196L TOP

[0061] MMLV_M63 GCCCCAGGGCTTTAAAAACAGCCATACATTGTTCGATGAAGCACTTCACCGT

[0062] P196H TOP

[0063] MMLV_M63 TAACCAAGCCCTTTGAGCTGTTCTCTGATGAAAAACAGGGATATGCAAAAGGA V370S TOP

[0064] MMLV_M63 CCCTTTGAGCTGTTCGTTGATGAAAAATTAGGATATGCAAAAGGAGTATTAACCCAA Q374L TOP

[0065] MMLV_M63 GCTGTTCGTTGATGAAAAACAGGGAATTGCAAAAGGAGTATTAACCCAAAAGTTAGGCCCGT Y376I TOP

[0066] MMLV_M63 GATGAAAAACAGGGATATGCAAAAGGATCTTTAACCCAAAAGTTAGGCCCGTGGCGT V380S TOP

[0067] MMLV_M63 CCTGTTGCTTACTTGAGTAAAAAATTGGATTTAGTCGCAGCAGGATGGCCACCGT P401LTOP

[0068] MMLV_M63 CAATTGCCGTTTTGACAAAGGATGCATCTAAGTTGACGATGGGTCAACCCTT

[0069] G424S TOP

[0070] MMLV_M63 GTTTTGACAAAGGATGCAGGTAAGTTGTGTATGGGTCAACCCTTAAACATCTTGGCT T427C TOP

[0071] MMLV_M63 AGTTGACGATGGGTCAACCCTTAACAATCTTGGCTCCACATGCTGTAGA

[0072] V433T TOP

[0073] MMLV_M63 CGATGGGTCAACCCTTAAACATCCATGCTCCACATGCTGTAGAAGCGT

[0074] K435H TOP

[0075] MMLV_M63 GATGGGTCAACCCTTAAACATCTTGTCTCCACATGCTGTAGAAGCGTTAGT

[0076] A436S TOP

[0077] MMLV_M63 ACTATCAGGCGCTTCTGCTTGATAACGATCGTGTACAATTTGGACCAGTTGT

[0078] T467N TOP

[0079] MMLV_M63 CTTGATACGGATCGTGTACAATTTGGATCTGTTGTAGCTTTGAATCCAGCTACTTTGC P474S TOP

[0080] MMLV_M63 GATCGTGTACAATTTGGACCAGTTGTATATTTGAATCCAGCTACTTTGCTTCCCCT A477Y TOP

[0081] MMLV_M63 GCTTCCCCTTCCAGAAGAAGGAACACAGCACAATTGTTTAGATATTCTGGCCGA L491T TOP

[0082] MMLV_M63 GACTTCAGCACAATTGTTTAGATATTCTGCAAGAGGCACATGGGACGCGCCCT A500Q TOP

[0083] MMLV_M63 CAGCACAATTGTTTAGATATTCTGGCCGAGTAACAAAGCCCGAAAGGAAGCTGAG A502STOP

[0084] TOP MMLV_M63 GTTTAGATATTCTGGCCGAGGCACGTGGGACGCGCCCTGATTTGA

[0085] H503R TOP

[0086] MMLV_M63 TAGATATTCTGGCCGAGGCACATGCGACGCGCCCTGATTTGACGGA

[0087] G504A TOP

[0088] MMLV_M63 CTGGCCGAGGCACATGGGTCTCGCCCTGATTTGACGGATCAGC

[0089] T505S TOP

[0090] MMLV_M63 CTGATGCCGACCATACATGGTATTTAGGCGGCAGTAGTCTTCTTCAAGA

[0091] T523L TOP

[0092] MMLV_M63 GATGCCGACCATACATGGTATACTAACGGCAGTAGTCTTCTTCAAGAGGGGCA G524N TOP

[0093] MMLV_M63 CATGGTATACTGGCGGCAGTAGTGTACTTCAAGAGGGGCAACGCAAGGCGGGA L528V TOPMMLV_M63 CATGGTATACTGGCGGCAGTAGTTATCTTCAAGAGGGGCAACGCAAGGCGGGA

[0094] L528Y TOP

[0095] MMLV_M63 ATACTGGCGGCAGTAGTCTTCTTGTAGAGGGGCAACGCAAGGCGGGA

[0096] Q530V TOP

[0097] MMLV_M63 CTGGCGGCAGTAGTCTTCTTCAATCTGGGCAACGCAAGGCGGGA

[0098] E531S TOP

[0099] MMLV_M63 CTTCAAGAGGGGCAACGCAAGTAACAAAGCCCGAAAGGAAGCTGAG

[0100] A536STOP

[0101] TOP MMLV_M63 GAGACCGAAGTTATCTGGGCCTGGGCGTTACCCGCGGGAACAT

[0102] K55OW TOP

[0103] MMLV_M63 TTGAATGTCTACACCAACTCACGTTATACATTTGCAACAGCGCATTGGCATGGCGA

[0104] A587T TOP

[0105] MMLV_M63 CGTTATGCTTTTGCAACAGCGCATTTACATGGCGAAATTTACCGCCGCCGT

[0106] W593L TOP

[0107] MMLV_M63 CCGCCGCCGTGGTCTGGGGACTAGTGAGGGTAAGGAAATTAAAAATAAAGA

[0108] L604G TOP

[0109] MMLV_M63 GCCGTGGTCTGCTGACTAGTTCTGGTAAGGAAATTAAAAATAAAGATGAGATTCTTGC

[0110] E607S TOP

[0111] MMLV_M63 GACTAGTGAGGGTAAGGAAATTAAAAATAAACCCGAGATTCTTGCGTTGTTAAAAGCTTTATTC D615P TOP

[0112] MMLV_M63 GACTAGTGAGGGTAAGGAAATTAAAAATAAACGTGAGATTCTTGCGTTGTTAAAAGCTTTATTC D615R TOP

[0113] MMLV_M63 TGAGGGTAAGGAAATTAAAAATAAAGATGAGGTACTTGCGTTGTTAAAAGCTTTATTCTTACC I617V TOP

[0114] MMLV_M63 GGGTAAGGAAATTAAAAATAAAGATGAGATTCTTTTATTGTTAAAAGCTTTATTCTTACCAAAACGCCT A619L TOP

[0115] MMLV_M63 GGGTAAGGAAATTAAAAATAAAGATGAGATTCTTCGTTTGTTAAAAGCTTTATTCTTACCAAAACGCCT A619R TOP

[0116] MMLV_M63 TGCGTTGTTAAAAGCTTTATTCTTACCAAAAACACTTTCGATCATTCATTGCCCGGGGCA

[0117] R629T TOP

[0118] MMLV_M63 ACCCCTTAACAAAAACAGGGACGTCTTTCAACTGGGGGCCAGACC

[0119] L333S TOP

[0120] MMLV_M63 ACCCCTTAACAAAAACAGGGACGGAGTTCAACTGGGGGCCAGACC

[0121] L333E TOP

[0122] MMLV M63 CTTTGCTTCCCCTTCCAGAAGAAGCGCTTCAGCACAATTGTTTAGATATTCTGGCCGA

[0123] G490A TOP

[0124] MMLV M63 CTTTGCTTCCCCTTCCAGAAGAAAACCTTCAGCACAATTGTTTAGATATTCTGGCCGA

[0125] G490N TOP

[0126] MMLV M63 CAAAAGGGTCACTCAGCGGAGGTACGTGGAAACCGTATGGCGGA

[0127] A646V TOP

[0128] MMLV_M63 GGTATTTGCTGAAAGAAGGTCAACGTCGTATTACTGATGCGCGTAAGGAGAC

[0129] W279R TOP

[0130] MMLV_M63 TTGGATTACTGATGCGCGTAAGGAGGATGTAATGGGGCAGCCTACGCC

[0131] T287D TOP

[0132] MMLV_M63 CGTTATGCTTTTGCAACAGCGCATTTACATGGCGAAATTTACCGCCGCCGT

[0133] W593L TOP

[0134] MMLV_M63 GTTCTTTTGATGTCTCATGTAAACGATGGGGATCCTCAATATTTAAAGTCATATGGCTGC

[0135] E7P BTMMMLV_M63 GTCGGGTTCTTTTGATGTCTCATGTAATAAATGCTCATCCTCAATATTTAAAGTCATATGG R9L BTM

[0136] MMLV_M63 GTCGGGTTCTTTTGATGTCTCATGTAACGCATGCTCATCCTCAATATTTAAAGTCATATGG R9A BTM

[0137] MMLV_M63 AGGCCTGATAACAGATTATAAGGATTAGGATAAGTCGGATGGATGTCTTCTACGC V129Y BTM

[0138] MMLV_M63 GCGATGGGGGCAGGCCTGACGCCAGATTATAAGGATTAGGTACAGTCGG L136ABTM

[0139] MMLV_M63 GCGATGGGGGCAGGCCGAATAACAGATTATAAGGATTAGGTACAGTCG

[0140] S137F BTM

[0141] MMLV_M63 ATTGGTGCGATGGGGGCAGAGATGATAACAGATTATAAGGATTAGGTACAGTC G138S BTM

[0142] MMLV_M63 TTGGGTGCAGACGCAGGCATACGAACGCGTCTTTCAAGTCTAATACTG

[0143] F156V BTM

[0144] MMLV_M63 ACGTTGGGTGCAGACGCAGCGCAAAGAACGCGTCTTTCAAGTCTAATAC

[0145] C157A BTM

[0146] MMLV_M63 ACCCGAAATTCCCATTTCAGGATCCTCCCATTCGAACGCAAACAGCGG

[0147] R173EBTM

[0148] MMLV_M63 ACGGTGAAGTGCTTCATCGAACAATGTCGCGCTGTTTTTAAAGCCCTGGGGC P196ABTM

[0149] MMLV_M63 ACGGTGAAGTGCTTCATCGAACAATGTTAAGCTGTTTTTAAAGCCCTGGGGC P196L BTM

[0150] MMLV_M63 ACGGTGAAGTGCTTCATCGAACAATGTATGGCTGTTTTTAAAGCCCTGGGGC P196H BTM

[0151] MMLV_M63 TCCTTTTGCATATCCCTGTTTTTCATCAGAGAACAGCTCAAAGGGCTTGGTTA V370S BTM

[0152] MMLV_M63 TTGGGTTAATACTCCTTTTGCATATCCTAATTTTTCATCAACGAACAGCTCAAAGGG Q374L BTM

[0153] MMLV_M63 ACGGGCCTAACTTTTGGGTTAATACTCCTTTTGCAATTCCCTGTTTTTCATCAACGAACAGC Y376I BTM

[0154] MMLV_M63 ACGCCACGGGCCTAACTTTTGGGTTAAAGATCCTTTTGCATATCCCTGTTTTTCATC V380S BTM

[0155] MMLV_M63 ACGGTGGCCATCCTGCTGCGACTAAATCCAATTTTTTACTCAAGTAAGCAACAGG P401L BTM

[0156] MMLV_M63 AAGGGTTGACCCATCGTCAACTTAGATGCATCCTTTGTCAAAACGGCAATTG G424S BTM

[0157] MMLV_M63 AGCCAAGATGTTTAAGGGTTGACCCATACACAACTTACCTGCATCCTTTGTCAAAAC T427C BTM

[0158] MMLV_M63 TCTACAGCATGTGGAGCCAAGATTGTTAAGGGTTGACCCATCGTCAACT

[0159] V433T BTM

[0160] MMLV_M63 ACGCTTCTACAGCATGTGGAGCATGGATGTTTAAGGGTTGACCCATCG

[0161] K435H BTM

[0162] MMLV_M63 ACTAACGCTTCTACAGCATGTGGAGACAAGATGTTTAAGGGTTGACCCATC

[0163] A436S BTM

[0164] MMLV_M63 ACAACTGGTCCAAATTGTACACGATCGTTATCAAGCAGAAGCGCCTGATAGT T467N BTM

[0165] MMLV_M63 GCAAAGTAGCTGGATTCAAAGCTACAACAGATCCAAATTGTACACGATCCGTATCAAG P474S BTM

[0166] MMLV_M63 AGGGGAAGCAAAGTAGCTGGATTCAAATATACAACTGGTCCAAATTGTACACGATC A477Y BTMMMLV_M63 TCGGCCAGAATATCTAAACAATTGTGCTGTGTTCCTTCTTCTGGAAGGGGAAGC

[0167] L491T BTM

[0168] MMLV_M63 AGGGCGCGTCCCATGTGCCTCTTGCAGAATATCTAAACAATTGTGCTGAAGTC

[0169] A500Q BTM

[0170] MMLV_M63 CTCAGCTTCCTTTCGGGCTTTGTTACTCGGCCAGAATATCTAAACAATTGTGCTG

[0171] A502STOP

[0172] BTM MMLV_M63 TCAAATCAGGGCGCGTCCCACGTGCCTCGGCCAGAATATCTAAAC

[0173] H503R BTM

[0174] MMLV_M63 TCCGTCAAATCAGGGCGCGTCGCATGTGCCTCGGCCAGAATATCTA

[0175] G504A BTM

[0176] MMLV_M63 GCTGATCCGTCAAATCAGGGCGAGACCCATGTGCCTCGGCCAG

[0177] T5O5S BTM

[0178] MMLV_M63 TCTTGAAGAAGACTACTGCCGCCTAAATACCATGTATGGTCGGCATCAG

[0179] T523L BTM

[0180] MMLV_M63 TGCCCCTCTTGAAGAAGACTACTGCCGTTAGTATACCATGTATGGTCGGCATC

[0181] G524N BTM

[0182] MMLV_M63 TCCCGCCTTGCGTTGCCCCTCTTGAAGTACACTACTGCCGCCAGTATACCATG

[0183] L528V BTM

[0184] MMLV_M63 TCCCGCCTTGCGTTGCCCCTCTTGAAGATAACTACTGCCGCCAGTATACCATG

[0185] L528Y BTM

[0186] MMLV_M63 TCCCGCCTTGCGTTGCCCCTCTACAAGAAGACTACTGCCGCCAGTAT

[0187] Q530V BTM

[0188] MMLV_M63 TCCCGCCTTGCGTTGCCCAGATTGAAGAAGACTACTGCCGCCAG

[0189] E531S BTM

[0190] MMLV_M63 CTCAGCTTCCTTTCGGGCTTTGTTACTTGCGTTGCCCCTCTTGAAG

[0191] A536STOP

[0192] BTM MMLV_M63 ATGTTCCCGCGGGTAACGCCCAGGCCCAGATAACTTCGGTCTC

[0193] K55OW

[0194] BTM MMLV_M63 TCGCCATGCCAATGCGCTGTTGCAAATGTATAACGTGAGTTGGTGTAGACATTCAA

[0195] A587T BTM

[0196] MMLV_M63 ACGGCGGCGGTAAATTTCGCCATGTAAATGCGCTGTTGCAAAAGCATAACG

[0197] W593L BTM

[0198] MMLV_M63 TCTTTATTTTTAATTTCCTTACCCTCACTAGTCCCCAGACCACGGCGGCGG

[0199] L604G BTM

[0200] MMLV_M63 GCAAGAATCTCATCTTTATTTTTAATTTCCTTACCAGAACTAGTCAGCAGACCACGGC

[0201] E607S BTM

[0202] MMLV_M63 GAATAAAGCTTTTAACAACGCAAGAATCTCGGGTTTATTTTTAATTTCCTTACCCTCACTAGTC D615PBTM

[0203] MMLV_M63 GAATAAAGCTTTTAACAACGCAAGAATCTCACGTTTATTTTTAATTTCCTTACCCTCACTAGTC D615R BTM

[0204] MMLV_M63 GGTAAGAATAAAGCTTTTAACAACGCAAGTACCTCATCTTTATTTTTAATTTCCTTACCCTCA I617V BTM

[0205] MMLV_M63 AGGCGTTTTGGTAAGAATAAAGCTTTTAACAATAAAAGAATCTCATCTTTATTTTTAATTTCCTTACCC A619L BTM

[0206] MMLV_M63 AGGCGTTTTGGTAAGAATAAAGCTTTTAACAAACGAAGAATCTCATCTTTATTTTTAATTTCCTTACCC A619R BTM108 MMLV_M63 TGCCCCGGGC AATGAATG ATCGAAAGTGTTTTTGGTAAGAATAAAGCTTTTAAC AACGC A R629T BTM

[0207] 109 MMLV_M63 GGTCTGGCCCCCAGTTGAAAGACGTCCCTGTTTTTGTTAAGGGGT

[0208] L333S BTM

[0209] 110 MMLV_M63 GGTCTGGCCCCCAGTTGAACTCCGTCCCTGTTTTTGTTAAGGGGT

[0210] L333E BTM

[0211] 111 MMLV M63 TCGGCCAGAATATCTAAACAATTGTGCTGAAGCGCTTCTTCTGGAAGGGGAAGCAAAG G490A SDM

[0212] BTM

[0213] 112 MMLV M63 TCGGCCAGAATATCTAAACAATTGTGCTGAAGGTTTTCTTCTGGAAGGGGAAGCAAAG G490N SDM

[0214] BTM

[0215] 113 MMLV M63 TCCGCCATACGGTTTCCACGTACCTCCGCTGAGTGACCCTTTTG

[0216] A646V SDM

[0217] BTM

[0218] 114 MMLV_M63 GTCTCCTTACGCGCATCAGTAATACGACGTTGACCTTCTTTCAGCAAATACC

[0219] W279R

[0220] BTM

[0221] 115 MMLV_M63 GGCGTAGGCTGCCCCATTACATCCTCCTTACGCGCATCAGTAATCCAA

[0222] T287D BTM

[0223] 116 MMLV_M63 ACGGCGGCGGTAAATTTCGCCATGTAAATGCGCTGTTGCAAAAGCATAACG

[0224] W593L BTM

[0225] Example 2 — Preparation of Reverse Transcriptase Mutants for Evaluation of Activity and Thermostability.

[0226] The data presented in Tables 2-14 was generated by in vitro assay of purified recombinant protein. All MMLV variants were generated in substantially the same manner and as is outlined herein.

[0227] A colony with the appropriate strain was used to inoculate 1 mL of TB media in a 96-well deep well plate. The culture was provided with kanamycin at a concentration of 0.05 mg / mL and grown at 37 °C. When the culture reached an OD of approximately 0.8, the plate was cooled on ice for 5 minutes. A 5 pL volume of 100 mM IPTG was added to each culture to induce protein expression, followed by incubation at 18 °C for 23 hours. Following induction, cells were harvested by centrifugation at 4,700 rpm for 10 minutes to produce a cell pellet. Cell pellets were produced in the aforementioned example for each examined MMLV RTase variant.

[0228] Each resultant cell pellet was resuspended in a lysis buffer containing 50 mM NaPO4, 5% glycerol, 300 mM NaCl, and 10 mM imidazole. Resuspended cells were lysed by the addition of lx BugBuster® (Millipore Sigma, Cat #70921) and incubation on an end-over-end mixer for 15 minutes at room temperature. Each lysate was subsequently centrifuged at 4,700 rpm for 10 minutes and at a temperature of 4 °C to remove cell debris.The cleared lysates were applied to a HisPur™ Ni-NTA spin plate (ThermoFisher, Cat #88230). The resin was equilibrated with Screening His-Bind buffer (50 mM NaPO4, pH 7.8, 5% glycerol, 300 mM NaCl, and 10 mM imidazole) and cleared lysate was subsequently loaded. Samples were washed three times with Screening His-Wash buffer (50 mM NaPO4, pH 7.8, 5% glycerol, 300 mM NaCl, and 25 mM imidazole) and eluted using Screening His-Elution buffer (50 mM NaPO4, pH 7.8, 5% glycerol, 300 mM NaCl, and 250 mM imidazole). Purified proteins were normalized to a set concentration of 130 mM for testing purposes.

[0229] Example 3 — Evaluation of MMLV Reverse Transcriptase Single Mutants.

[0230] The ability of each variant of the M63 variant base construct to synthesize cDNA from purified total Universal RNA (Agilent) was compared to purified protein having the sequence of SSII (Invitrogen; SEQ ID NO: 276) and the M63 variant base construct. In particular, MMLV RTase activity was assayed by random hexamer priming using a standard two-step cDNA synthesis. Thermostability was assessed by varying the assay temperature. Protein having the sequence of SSII (Invitrogen) was prepared according to the method given in Example 2 using BL21(DE3) transformed with a plasmid containing SEQ ID NO: 118, and is referred to herein as “MMLV-II.”

[0231] RTase s (2 pL 130 nM or 13 nM) were added to a reaction mixture containing RNA (50 ng), dNTPs (100 pM), random hexamer primers (5 ng / pL), first strand synthesis buffer (1x, 50 mM Tris-HCl, pH 8.3, 75 mM KC1, 3 mM MgCl2, 10 mM DTT), and SuperaselN (ThermoFisher; 0.17 U / pL) in a 50 pL volume. The reaction was allowed to proceed at a target temperature for a duration of 15 minutes, followed by incubation at 80 °C for 10 minutes. Multiple iterations of the two-step protocol were performed to vary the target temperature. As will be discussed in more detail subsequent, reverse transcription by random hexamer priming was performed at 50 °C, 55 °C, 60 °C, or 65 °C.

[0232] Cycle threshold (Ct) values describing the cDNA synthesis activity of the RTase mutants were quantified by qPCR amplification using an assay that identified the hypoxanthine phosphoribosyltransferase (HPRT) gene or the serine and arginine rich splicing factor 9 (SFRS9) gene in human cells. Both HPRT and SFRS9 were assayed for each MMLV RTase variant. An assay master mix including Integrated DNA Technologies PrimeTime® Gene Expression Master Mix (GEM) was prepared and used to prepare each assay. The master mix also included primers and probes for HPRT and SFRS9, for which sequence information is presented in Table 2. In the sequences listed in the “Primer / ProbeSequence (5’ - 3’)” column, “5 SUN” refers to a 5’ SUN (Integrated DNA Technologies) modification, “ZEN” refers to the ZEN internal quencher (Integrated DNA Technologies), “3IABkFQ” refers to 3' Iowa Black® FQ (Integrated DNA Technologies) modification, and “56-FAM” refers to a 5' 6-FAM (Fluorescein; Integrated DNA Technologies) modification. In particular, the assay master mix included GEM (1x), HPRT and SFRS9 primer sets (500 nM each, Table 2), and HPRT and SFRS9 probes (250 nM each, Table 2).

[0233] The assay master mix and synthesized cDNA (i.e., from the prior reaction) were mixed at a 10:1 ratio for a final volume of 20 µL. qPCR reactions proceeded on a QuantStudio7 Flex qPCR machine (ThermoFisher) for 40 cycles using the following cycle conditions: 95 °C hold for 3 minutes, 95 °C for 15 seconds, and 60 °C for one minute.

[0234] TABLE 2

[0235] Sequences of primers and probes used for qPCR assays.

[0236] Seq Primer

[0237] ID Name Primer / Probe Sequence (5’ - 3’)

[0238] 433 Hs GTCGAGTATCTCAGAAAAGAAGACA

[0239] SFRS9

[0240] Forward

[0241] Primer

[0242] 434 Hs CTCGGATGTAGGAAGTTTCACC

[0243] SFRS9

[0244] Reverse

[0245] Primer

[0246] 435 Hs / 5SUN / ATGCCCTGC / ZEN / GTAAACTGGATGACA / 3IABkFQ / SFRS9

[0247] Probe - SUN

[0248] 436 Hs GACTTTGCTTTCCTTGGTCAG

[0249] HPRT

[0250] Forward

[0251] Primer

[0252] 437 Hs GGCTTATATCCAACACTTCGTG

[0253] HPRT

[0254] Reverse

[0255] Primer

[0256] 438 Hs 156- HPRT FAM / ATGGTCAAG / ZEN / GTCGCAAGCTTGCTGGT / 3IABkFQ / Probe - FAM

[0257] A646V, G490A, E333E, K435H, R159P, R159S, R629M and T287D mutants were generated by site directed mutagenesis according to the method described subsequently with respect to Example 1. The amino acid sequences of the above-indicatedmutants as well as MMLV-II and the M63 variant MMLV RTase are provided in Table 3, below.

[0258] _ TABLE 3 _

[0259] MMLV RTase

[0260] Variant Name SEQ ID _ Amino Acid Sequence (N - C) _

[0261] A646V 432 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQETWTRLPQGFKNSPTLFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TLGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTLFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKLGPWRRPVAY ESKKLDPVAAGWPPCERMVAAIAVETKDAGKLTMGQ PENIEAPHAVEAEVKQPPDRWLSNARMTHYQAEEEDT DRVQFGPVVAENPATLEPEPEEGEQHNCEDIEAEAHGT RPDETDQPEPDADHTWYTGGSSEEQEGQRKAGAAVTT ETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI EAEEKAEFEPKRLSIIHCPGHQKGHSAEVRGNRMADQA ARKAAITETPDTSTLEIENSSPYTSEHF

[0262] G490A 430 MGSSHHHHHHSSGEVPRGSHMTENIEDEHRLHETSKEP DVSEGSTWLSDFPQAWAETGGMGEAVRQAPEIIPEKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGIEVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNEESGEPPSHQWYTVEDEKDAFFCERLHPTSQPEFAF EWRDPEMGISGQETWTRLPQGFKNSPTLFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TLGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTLFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKLGPWRRPVAY ESKKLDPVAAGWPPCERMVAAIAVETKDAGKLTMGQ PENIEAPHAVEAEVKQPPDRWLSNARMTHYQAEEEDT DRVQFGPVVAENPATLEPEPEEAEQHNCEDIEAEAHGT RPDETDQPEPDADHTWYTGGSSEEQEGQRKAGAAVTT ETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGEETSEGKEIKNKDEI EAEEKAEFEPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLEIENSSPYTSEHF

[0263] L333E 428 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0264] K435H 429 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNIHAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0265] R159P 425 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLPLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0266] R159S 426 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLSLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0267] R629M 431 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKMLSIIHCPGHQKGHSAEARGNRMADQ AARKAAITETPDTSTLLIENSSPYTSEHF

[0268] T287D 427 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0269] M63 Variant 277 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF MMLV-II 276 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPW NTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLT EARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0270] The two-step reactions for the MMLV variants were analyzed and reported by Ct value. Table 4, Table 5, Table 6, and Table 7 report Ct values for 50 °C, 55 °C, 60 °C, and 65 °C, respectively, with an RTase concentration of 5.2 nM. Table 8 reports Ct values at 65 °C with an RTase concentration of 0.52nM.

[0271] All single mutants provided in Table 3 (SEQ IDs: 425-432) showed increased activity relative to both the M63 variant and MMLV-II at 50 °C, 55 °C, 60 °C, and 65 °C (at 5.2 nM RTase) for both assay targets (i.e., HPRT and SFRS9). The A646V, G490A, L333E, K435, R159P, R159S, and T287D mutants also showed increased activity at 65 °C by the assay for SFRS9, while the R629M mutant showed increased activity relative to MMLV-II and comparable activity relative to the M63 variant. At 65 °C and using the lower enzyme RTase concentration the A646V, G490A, R159P, R159S, T287D mutants showed increased activity relative to both the M63 variant and MMLV-II for both gene assays. At the lower concentration of RTase (i.e., 0.52 nM) MMLV-II also showed increased activity relative to the M63 variant according to the assay for SFRS9. Moreover, the M63 variant also includes a mutation at position 435 (K435L). Notably, the K435H mutant shows increased activity relative to the M63 variant at 50 °C, 55 °C, 60 °C, and 65 °C.TABLE 4

[0272] Two-Step cDNA synthesis by mutants of modified MMLV RTase. Data was generated via qPCR assay for HPRT OR SFRS9, as indicated by the “Target Name” column. Data is reported as Ct values. Reaction temperatures were 50 °C and the indicated MMLV RTase variant was at a concentration of 5.2 nM.

[0273] Target Ct Ct MMLV RTase Variant Name Mean SD HPRT 29.32 0.04 SFRS9 28.54 0.02 G490A HPRT 29.74 0.07

[0274] SFRS9 28.95 0.06 HPRT 29.18 0.05 SFRS9 28.43 0.03 HPRT 29.33 0.36 SFRS9 28.63 0.30 HPRT 29.37 0.07 SFRS9 28.75 0.05 HPRT 29.77 0.03 SFRS9 29.03 0.05 R629M HPRT 29.85 0.03

[0275] SFRS9 29.08 0.01 T287D HPRT 28.97 0.06

[0276] SFRS9 28.27 0.03+HPRT 30.02 0.13M63 Va™"<SFRS9 Q o8

[0277]

[0278] MMLV-II HPRT 39.48 0.73

[0279] SFRS9 37.84 0.56

[0280] _ TABLE 5 _

[0281] Two-Step cDNA synthesis by triple and quadruple mutants of modified MMLV RTase. Data was generated via qPCR assay for HPRT OR SFRS9, as indicated by the “Target Name” column. Data is reported as Ct values.

[0282] Reaction temperatures were 55 °C and the indicated MMLV RTase variant was at a concentration of 5.2 nM.

[0283] Target Ct Ct RTase Name Mean SD HPRT 29.627 0.057 A646V

[0284] SFRS9 28.983 0.011 G490A HPRT 30.208 0.113

[0285] SFRS9 29.497 0.062 HPRT 29.583 0.064 L333E

[0286] SFRS9 28.999 0.023HPRT 29.985 0.334 SFRS9 29.369 0.349 HPRT 30.109 0.064 SFRS9 29.544 0.029 R159S HPRT 30.330 0.053

[0287]

[0288] SFRS9 29.565 0.052 R629MHPRT 30’412°’021

[0289] SFRS9 29.795 0.080 T287DHPRT 29515°’°25

[0290] SFRS9 29.156 0.080+HPRT 30.785 0.061 M63 Variant SFRS9 30.224 0.047

[0291] HPRT 39.638 0.513MMLV’nSFRS9 38.546 0.533

[0292] _ TABLE 6 _

[0293] Two-Step cDNA synthesis by triple and quadruple mutants of modified MMLV RTase. Data was generated via qPCR assay for HPRT OR SFRS9, as indicated by the “Target Name” column. Data is reported as Ct values. Reaction temperatures were 60 °C and the indicated MMLV RTase variant was at a concentration of 5.2 nM.

[0294] Target Ct Ct MMLV RTase Variant Name Mean SD A646V HPRT 30.40 0.13 A646V

[0295] SFRS9 29.80 0.04 G490A HPRT 31.23 0.13

[0296] SFRS9 30.75 0.06 L333E HPRT 30.43 0.12

[0297] SFRS9 29.91 0.06 HPRT 31.08 0.25 SFRS9 30.35 0.40 HPRT 30.85 0.06 SFRS9 30.56 0.10 HPRT 31.16 0.04 SFRS9 30.78 0.07 R629M HPRT 31.41 0.18 SFRS9 31.13 0.11 T287DHPRT 30’49°’17SFRS9 30.04 0.09. HPRT 31.54 0.32 M63 Variant „ _ _ _ _

[0298] SFRS9 31.19 0.12

[0299]

[0300] MMLV-II HPRT 40.00 0.00SFRS9 38.31 0.56

[0301] _ TABLE 7 _

[0302] Two-Step cDNA synthesis by triple and quadruple mutants of modified MMLV RTase. Data was generated via qPCR assay for HPRT OR SFRS9, as indicated by the “Target Name” column. Data is reported as Ct values. Reaction temperatures were 65 °C and the indicated MMLV RTase variant was at a concentration of 5.2 nM.

[0303] Target Ct Ct RTase Name Mean SD HPRT 30.29 0.11A646VSFRS9 29.65 0.06 G490AHPRT 30’89 0-USFRS9 30.29 0.06 HPRT 30.43 0.02

[0304]

[0305] SFRS9 29.85 0.13 HPRT 31.27 0.28

[0306]

[0307] SFRS9 30.65 0.36 HPRT 30.79 0.11

[0308]

[0309] SFRS9 30.19 0.09159SHPRT 30.89 0.08

[0310]

[0311] SFRS9 30.28 0.04 R629MHPRT 31 49 0 10

[0312] SFRS9 31.01 0.10 HPRT 30.35 0.04 SFRS9 29.80 0.06. HPRT 31.63 0.12

[0313]

[0314] ananSFRS9 30.99 0.07

[0315] HPRT 39.50 0.71MMLV’nSFRS9 37.85 0.07

[0316] _ TABLE 8 _

[0317] Two-Step cDNA synthesis by triple and quadruple mutants of modified MMLV RTase. Data was generated via qPCR assay for HPRT OR SFRS9, as indicated by the “Target Name” column. Data is reported as Ct values. Reaction temperatures were 65 °C and the indicated MMLV RTase variant was at a concentration of 0.52 nM.

[0318] Target Ct Ct RTase Name Mean SD HPRT 34.42 0.34 A646V

[0319] SFRS9 34.72 0.21 G490A HPRT 35.59 1.86SFRS9 34.84 0.28

[0320] HPRT 39.23 1.08

[0321]

[0322] SFRS9 37.42 0.80

[0323] HPRT 39.08 1.30

[0324]

[0325] SFRS9 37.86 1.19

[0326] HPRT 35.87 0.71

[0327]

[0328] SFRS9 34.78 0.24

[0329] HPRT 35.94 0.59

[0330]

[0331] SFRS9 36.21 0.74

[0332] R629MHPRT 38 50 1 06

[0333] SFRS9 37.87 1.01

[0334] T287DHPRT 35 74 0 23

[0335] SFRS9 35.67 0.56

[0336] . HPRT 36.74 0.49

[0337] M63 Variant „ _

[0338] SFRS9 36.79 0.56

[0339] HPRT 36.85 0.74

[0340] MMLV‘nSFRS9 36.02 0.33

[0341] Example 4 — Evaluation of MMLV Reverse Transcriptase Combination Mutants.

[0342] Various combination mutants of the single mutants examined in Example 3 were generated to examine their activity and thermostability. In particular, T287D / L333E / K435H, T287D / L333E / G490, T287D / L333E / R629M, T287D / L333E / A646V, T287D / L333E / R159P, T287D / L333E / K435H / G490A, T287D / L333E / K435H / R629M, T287D / L333E / K435H / A646V, R159P / T287D / L333E / K435H, T287D / L333E / G490A / R629M, T287D / L333E / G490A / A646V, R159P / T287D / L333E / G490A, T287D / L333E / R629M / A646V, R159P / T287D / L333E / R629M, and R159P / T287D / L333E / A646V variants were generated by site directed mutagenesis according to the method described subsequently with respect to Example 1. All mutations were made to the M63 variant base sequence. The amino acid sequences of the aboveindicated mutants as well as MMLV-II and the M63 variant MMLV RTase are provided in Table 9, below.

[0343] _ TABLE 9 _

[0344] MMLV RTase

[0345] Variant Name SEQ ID _ Amino Acid Sequence (N - C) _

[0346] M205 – M63 + 278 MGSSHHHHHHSSGEVPRGSHMTENIEDEHRLHETSKEP T287D / L333E / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT K435H STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQETWTRLPQGFKNSPTLFDEAEHRDEADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPA EGEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVA YESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMG QPENIHAPHAVEAEVKQPPDRWESNARMTHYQAEEED TDRVQFGPVVAENPATEEPEPEEGEQHNCEDIEAEAHG TRPDETDQPEPDADHTWYTGGSSEEQEGQRKAGAAVT TETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKE NVYTNSRYAFATAHWHGEIYRRRGEETSEGKEIKNKD EIEAEEKAEFEPKRESIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTEEIENSSPYTSEHF

[0347] M206 – M63 + 279 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEPT287D / L333E / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATG490A STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPWNTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEALQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPYTSEHF

[0348] M207 - M63 + 280 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / L333E / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT R629M STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKMLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0349] M208 - M63 + 281 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / L333E / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT A646V STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEVRGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0350] M209 - M63 + 282 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / L333E / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT R159P STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLPLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0351] M210-M63 + 283 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / L333E / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT K435H / G490A STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNIHAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEALQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0352] M211 -M63 + 284 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / L333E / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT K435H / R629M STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNIHAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKMLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF*

[0353] M212-M63 + 285 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / L333E / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT K435H / A646V STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNIHAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEVRGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0354] M213 -M63 + 286 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R159P / T287D / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT L333E / K435H STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLPLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNIHAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0355] M214-M63 + 287 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / L333E / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT G490A / R629M STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW

[0356] NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEALQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKMLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0357] M215 -M63 + 288 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / L333E / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT G490A / A646V STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEALQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEVRGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0358] M216-M63 + 289 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R159P / T287D / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT L333E / G490A STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLPLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEALQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0359] M217 -M63 + 290 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / L333E / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT R629M / A646V STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPWNTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLPLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKMLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0360] M218 -M63 + 291 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R159P / T287D / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT L333E / R629M STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLPLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKMLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0361] M219 -M63 + 292 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R159P / T287D / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT L333E / A646V STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLPLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEVRGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHFThe ability of each combination mutant to synthesize cDNA from purified total Universal RNA (Agilent) was compared to purified protein having the sequence of SSII (Invitrogen; SEQ ID NO: 276) and the M63 variant base construct. In particular, the two-step assay described in the discussion of Example 3 was used with target temperatures of 50 °C, 55 °C, 60 °C, 65 °C and a final concentration of reverse transcriptase of 5.2 nM. The two-step reactions for the MMLV variants were analyzed and reported by Ct value. Table 10 reports Ct values for each variant (including the M63 and MMLV-II variants) at each target temperature.

[0362] The T287D / L333E / K435H, T287D / L333E / G490, T287D / L333E / R629M, T287D / L333E / A646V, T287D / L333E / K435H / R629M, T287D / L333E / K435H / A646V, R159P / T287D / L333E / K435H, T287D / L333E / G490A / R629M, T287D / L333E / G490A / A646V, R159P / T287D / L333E / G490A, T287D / L333E / R629M / A646V, R159P / T287D / L333E / R629M, and R159P / T287D / L333E / A646V variants showed increased activity relative to both the M63 variant MMLV RTase and MMLV-II at all temperatures. The T287D / L333E / R159P and T287D / L333E / K435H / G490A variants showed comparable activity to the M63 variant RTase and improved activity over MMLV-II at all temperatures.

[0363] TABLE 10

[0364] Two-Step cDNA synthesis by triple and quadruple mutants of modified MMLV RTase. Data was generated via qPCR assay for HPRT OR SFRS9, as indicated by the “Target Name” column. Data is reported as

[0365] Ct values.

[0366] K jr v nT • cDNA Target Ct Ct MMLV RTase Variant „..&

[0367] Temp Name Mean SD

[0368] 50 °C HPRT 30.486 0.115

[0369] 55 °C HPRT 30.804 0.132

[0370] 60 °C HPRT 31.452 0.031 T9«7TV / T 65 °C

[0371] T287D / L333E / K435H

[0372]

[0373] 50 oc SHFPRRST

[0374] 93

[0375] 2812

[0376] 66

[0377] 59

[0378] 8°

[0379] 0 0494

[0380] 4

[0381] 55 °C SERS9 29.102 0.047

[0382] 60 °C SERS9 29.602 0.060

[0383] 65 °C SERS9 29.357 0.032

[0384] 50 °C HPRT 30.610 0.134

[0385] 55 °C HPRT 31.285 0.091

[0386] 60 °C HPRT 31.790 0.048

[0387] T T228877DD / / TL 333333LE / / GG449900AA6550°0CQ SHFPRRST932813%629°0 07341

[0388] 55 °C SFRS9 29.806 0.048

[0389] 60 °C SFRS9 30.190 0.182

[0390] 65 °C SFRS9 29.612 0.04650 °C HPRT 30.267 0.063

[0391] 55 °C HPRT 30.920 0.121 60 °C HPRT 31.646 0.096 T T022877DD / / TL Q3Q3Q3TE7 / / RPA692Q9AM46550° oCC SHFPRRST93208’48817200.004169

[0392] 55 °C SFRS9 29.158 0.038 60 °C SFRS9 29.873 0.050 65 °C SFRS9 29.170 0.019 50 °C HPRT 30.378 0.133 55 °C HPRT 30.985 0.029 60 °C HPRT 31.592 0.226 T D2fi877nD / / LT ^333E / / AA6^46AvV5650„ °Cc sHpPRRST932g1’2652Q8°Q-0Q7646

[0393] 55 °C SFRS9 29.375 0.063 60 °C SFRS9 29.982 0.170 65 °C SFRS9 29.331 0.084 50 °C HPRT 32.162 0.046 55 °C HPRT 32.870 0.309 60 °C HPRT 34.293 0.085 65 °C T287D / L333E / R159P50 oc SHFPRRST

[0394] 93330’787801°0’01792055 °C SFRS9 31.861 0.036 60 °C SFRS9 33.414 0.338 65 °C SFRS9 32.300 0.091 50 °C HPRT 32.486 0.151 55 °C HPRT 32.958 0.115 60 °C HPRT 34.279 0.273 65 °C HPRT 34533 0 143 T287D / L333E / K435H / G490ASFRS9

[0395] 55 °C SFRS9 31.482 0.065 60 °C SFRS9 32.755 0.083 65 °C SFRS9 32.626 0.147 50 °C HPRT 30.583 0.111 55 °C HPRT 30.974 0.017 60 °C HPRT 31.655 0.217 T287D / L333E / K435H / R629M 6550°oCc SHFPRRST93281-664843°o-0o5n2

[0396] 55 °C SFRS9 29.268 0.017 60 °C SFRS9 30.099 0.200 65 °C SFRS9 29.790 0.029 50 °C HPRT 30.050 0.074 55 °C HPRT 30.932 0.106 60 °C HPRT 31 316 0074 T287D / L333E / K435H / A646VHPRT 3t5110409

[0397] 50 °C SFRS9 28.181 0.035

[0398]

[0399] 55 °C SFRS9 29.133 0.02460 °C SFRS9 29.790 0.083

[0400] 65 °C SFRS9 29.665 0.084 50 °C HPRT 29.934 0.087 55 °C HPRT 30.652 0.091 60 °C HPRT 31.017 0.122 R159P / T287D / L333E / K435H 6550°oCc SHFPRRST93208’619941°Q-004843

[0401] 55 °C SFRS9 28.996 0.108 60 °C SFRS9 29.437 0.070 65 °C SFRS9 28.981 0.070 50 °C HPRT 30.323 0.075 55 °C HPRT 31.170 0.131 60 °C HPRT 31.541 0.164 T287D / L333E / G490A / R629M ™

[0402] 55 °C SFRS9 29.450 0.019 60 °C SFRS9 29.880 0.163 65 °C SFRS9 29.454 0.042 50 °C HPRT 30.219 0.085 55 °C HPRT 31.038 0.050 60 °C HPRT 31.557 0.039 65 °C HPRT 31 200 0053 T287D / L333E / G490A / A646VSFRS9

[0403] 55 °C SFRS9 29.572 0.058 60 °C SFRS9 29.961 0.160 65 °C SFRS9 29.483 0.031 50 °C HPRT 29.779 0.183 55 °C HPRT 30.765 0.102 60 °C HPRT 30.892 0.062 R R115599PP / / TT228877DD / / 1L 333333FE / / GG449900AA6550°oCc SHFPRRST93208’50587400’015661

[0404] 55 °C SFRS9 29.142 0.047 60 °C SFRS9 29.208 0.076 65 °C SFRS9 28.735 0.012 50 °C HPRT 30.028 0.110 55 °C HPRT 30.923 0.072 60 °C HPRT 31.068 0.101 65 °C HPRT 30771 0019 T287D / L333E / R629M / A646VSFRS9 2833g55 °C SFRS9 29.337 0.052 60 °C SFRS9 29.473 0.151 65 °C SFRS9 29.072 0.033 50 °C HPRT 30.191 0.144 55 °C HPRT 31 094 0077 R159P / T287D / L333E / R629MHPRT31490 0466

[0405]

[0406] 65 °C HPRT 30.900 0.06250 °C SFRS9 28.509 0.107

[0407] 55 °C SFRS9 29.525 0.064

[0408] 60 °C SFRS9 29.532 0.074

[0409] 65 °C SFRS9 29.300 0.085

[0410] 50 °C HPRT 30.299 0.093

[0411] 55 °C HPRT 30.907 0.028

[0412] 60 °C HPRT 31.528 0.164

[0413] 65 °C HPRT 31.046 0.043 R159P / T287D / L333E / A646VSFRS9

[0414] 55 °C SFRS9 29.407 0.010

[0415] 60 °C SFRS9 29.719 0.098

[0416] 65 °C SFRS9 29.458 0.132

[0417] 50 °C HPRT 31.422 0.091

[0418] 55 °C HPRT 31.712 0.070

[0419] 60 °C HPRT 32.411 0.047

[0420] . 65 °C HPRT 31.925 0.108

[0421]

[0422] M63 Variant50 °C SFRS9 29.208 0.005

[0423] 55 °C SFRS9 30.293 0.028

[0424] 60 °C SFRS9 30.760 0.160

[0425] 65 °C SFRS9 30.074 0.039

[0426] 50 °C HPRT 39.272 0.516

[0427] 55 °C HPRT 39.535 0.657

[0428] 60 °C HPRT 40.000 0.000 MMLV-II65 °C HPRT 40.000 0.000

[0429] V’ 50 °C SFRS9 36.402 0.495

[0430] 55 °C SFRS9 37.392 0.225

[0431] 60 °C SFRS9 36.189 0.523

[0432] 65 °C SFRS9 36.265 0.668

[0433] Example 5 — Evaluation of additional MMLV Reverse Transcriptase Single Mutants.

[0434] Various additional single mutants of the M63 variant base construct were generated to examine their activity and thermostability. In particular, A502STOP, A536STOP, A619L, C157A, D615P, D615R, E531S, E607S, E7P, F156V, G138S, G424S, G504A, H503R, I617V, K550W, L136A, K435H, L491T, L528V, L528Y, L604G, V433T, P196A, P196H, P196L, P401L, Q374L, Q530V, R173E, R9A, R9L, S137F, T427C, T467N, T505S, V129Y, V370S, V380S, and Y376I variants were generated by site directed mutagenesis according to the method described subsequently with respect to Example 1. All mutations were made to the M63 variant base sequence. The amino acid sequences of the above-indicated mutants are provided in Table 11, below.TABLE 11

[0435] MMLV RTase

[0436] Variant Name SEQ ID _ Amino Acid Sequence (N - C) _

[0437] M63 + 294 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP A502STOP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDT DRVQFGPVVAENPATEEPEPEEGEQHNCEDIEAE

[0438] M63 + 295 MGSSHHHHHHSSGEVPRGSHMTENIEDEHREHETSKEP A536STOP DVSEGSTWESDFPQAWAETGGMGEAVRQAPEIIPEKAT STPVSIKQYPMSREAREGIKPHIRREYDQGIEVPCQSPW NTPERPVKKPGTNDYRPVQDEREVNKRVEDIHPTVPNP YNEESGEPPSHQWYTVEDEKDAFFCEREHPTSQPEFAF EWRDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDT DRVQFGPVVAENPATEEPEPEEGEQHNCEDIEAEAHGT RPDETDQPEPDADHTWYTGGSSEEQEGQRK

[0439] M63 + A619L 296 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSEGSTWESDFPQAWAETGGMGEAVRQAPEIIPEKAT STPVSIKQYPMSREAREGIKPHIRREYDQGIEVPCQSPW NTPERPVKKPGTNDYRPVQDEREVNKRVEDIHPTVPNP YNEESGEPPSHQWYTVEDEKDAFFCEREHPTSQPEFAF EWRDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDT DRVQFGPVVAENPATEEPEPEEGEQHNCEDIEAEAHGT RPDETDQPEPDADHTWYTGGSSEEQEGQRKAGAAVTT ETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKEN VYTNSRYAFATAHWHGEIYRRRGEETSEGKEIKNKDEI EEEEKAEFEPKRESIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTEEIENSSPYTSEHF

[0440] M63 + C157A 297 MGSSHHHHHHSSGEVPRGSHMTENIEDEHREHETSKEP DVSEGSTWLSDFPQAWAETGGMGEAVRQAPEIIPEKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGIEVPCQSPW NTPERPVKKPGTNDYRPVQDEREVNKRVEDIHPTVPNP YNEESGEPPSHQWYTVEDEKDAFFAEREHPTSQPEFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0441] M63 +D615P 298 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKPEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0442] M63 +D615R 299 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKREI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0443] M63 + E531S 300 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDT DRVQFGPVVAENPATEEPEPEEGEQHNCEDIEAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQSGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0444] M63 + E607S 301 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSSGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0445] M63 + E7P 302 MTLNIEDPHRLHETSKEPDVSLGSTWLSDFPQAWAETG GMGLAVRQAPLIIPLKATSTPVSIKQYPMSREARLGIKP HIRRLYDQGILVPCQSPWNTPLRPVKKPGTNDYRPVQD LREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLK DAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQ GFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLA ATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQV KYLGYLLKEGQRWITDARKETVMGQPTPKTPRELREF LGKAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQ QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAK GVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVA AIAVLTKDAGKLTMGQPLNILAPHAVEALVKQPPDRW LSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEE GLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGGS SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAQLI ALTQALKMAEGKKLNVYTNSRYAFATAHWHGEIYRR RGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQK GHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPY TSEHF

[0446] M63 +F156V 303 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFVCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0447] M63 + G138S 304 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSSLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFE WRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLA DFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDA RKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMA APLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALG LPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTR PDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNV YTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEIL ALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0448] M63 + G424S 305 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDASKLTMGQP LNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTR PDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNV YTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEIL ALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0449] M63 + G504A 306 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHAT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0450] M63 + H503R 307 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEARGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0451] M63 + I617V 308 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDE VLALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQ AARKAAITETPDTSTLLIENSSPYTSEHF

[0452] M63 + K550W 309 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAWALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0453] M63 + L136A 310 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLASGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0454] M63 + K435H 311 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNIHAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0455] M63 + L491T 312 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGTQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0456] M63 + L528V 313 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSVLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0457] M63 + L528Y 314 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSYLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0458] M63 + L604G 315 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLGTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0459] M63 + V433T 316 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLTILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0460] M63 + P196A 317 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSATLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0461] M63 + P196H 318 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSHTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0462] M63 + P196L 319 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSLTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0463] M63 + P401L 320 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDLVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0464] M63 + Q374L 321 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKLGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0465] M63 + Q530V 322 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLVEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0466] M63 + R173E 323 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWEDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLA DFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDA RKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMA APLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALG LPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTR PDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNV YTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEIL ALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0467] M63 + R9A 324 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHALHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0468] M63 + R9L 325 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHLLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0469] M63 + S137F 326 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLFGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0470] M63 + T427C 327 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLCMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0471] M63 + T467N 328 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDN DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0472] M63 + T505S 329 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGS RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0473] M63 + V129Y 330 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTYPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0474] M63 + V370S 331 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFSDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0475] M63 + V380S 332 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGSLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0476] M63 + Y376I 333 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGIAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTR PDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNV YTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEIL ALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0477] The ability of each combination mutant to synthesize cDNA from purified total Universal RNA (Agilent) was compared to purified protein having the sequence of SSII (Invitrogen; SEQ ID NO: 276) and the M63 variant base construct. In particular, the two-step assay described in the discussion of Example 3 was used with target temperatures of 50 °C, 55 °C, 60 °C, 65 °C and a final concentration of reverse transcriptase of 5.2 nM. The two-step reactions for the MMLV variants were analyzed and reported by Ct value. Table 12 reports Ct values for each variant (including the M63 and MMLV-II variants) at 50 °C and 55 °C, and Table 13 Ct values for each variant (including the M63 and MMLV-II variants) at 60 °C and 65 °C.

[0478] As is shown in the data of Table 12 and Table 13, all mutants showed significantly improved activity as compared to the MMLV-II variant at all examined temperatures. Further, all mutants showed comparable or improved activity as compared to the M63 variant MMLV RTase. In particular, the R9A (SEQ ID NO: 324), C157A (SEQ ID NO: 297), R173E (SEQ ID NO: 323), V433T (SEQ ID NO: 316), T467N (SEQ ID NO: 328), A502STOP (SEQ ID NO: 294), and L528Y (SEQ ID NO: 314) variants showed significantly increased activity at multiple temperatures (as determined by at least one of the HPRT and SFRS9 assays) as compared to both M63 and MMLV-II variants. Notably, the M63 variant also includes a mutation to valine 433 (V433N). Advantageously, the V433T mutant shows improved activity relative to the M63 variant at 50 °C, 55 °C, 60 °C, and 65 °C. P

[0479] TABLE 12

[0480] Two-Step cDNA synthesis by single mutants of modified MMLV RTase. Data was generated via qPCR assay for HPRT OR SFRS9, as indicated by the “Target Name” column. Data is reported as Ct values.

[0481] cDNA Target Ct Ct MMLV RTase Variant

[0482] Temp Name Mean SD50 °C HPRT 31.156 0.178 55 °C HPRT 32.338 0.170

[0483]

[0484] A436S 50 °C SFRS9 30.158 0.084

[0485] 55 °C SFRS9 31.784 0.086 50 °C HPRT 29.704 0.108 A502STOP 55 °C HPRT 31.389 0.095

[0486] 50 °C SFRS9 29.302 0.079 55 °C SFRS9 30.827 0.093 50 °C HPRT 30.485 0.190 55 °C HPRT 31.401 0.092

[0487]

[0488] 50 °C SFRS9 29.966 0.057 55 °C SFRS9 31.340 0.150 50 °C HPRT 30.666 0.226A i r55 °C HPRT 31.714 0.175 An19T

[0489] 50 °C SFRS9 29.629 0.032 55 °C SFRS9 31.508 0.088 50 °C HPRT 29.816 0.096 55 °C HPRT 31.025 0.120

[0490]

[0491] C157A 50 °C SFRS9 29.103 0.115

[0492] 55 °C SFRS9 30.763 0.136 50 °C HPRT 30.876 0.073 55 °C HPRT 31.984 0.051

[0493]

[0494] D615P 50 °C SFRS9 29.864 0.006

[0495] 55 °C SFRS9 31.829 0.105 50 °C HPRT 31.402 0.105 55 °C HPRT 32.369 0.074

[0496]

[0497] D615R 50 °C SFRS9 30.204 0.084

[0498] 55 °C SFRS9 31.917 0.181 50 °C HPRT 30.812 0.137 55 °C HPRT 31.420 0.143 E531S 50 °C SFRS9 29.816 0.104

[0499] 55 °C SFRS9 30.951 0.181 50 °C HPRT 31.354 0.212 55 °C HPRT 31.934 0.062 E607S 50 °C SFRS9 30.019 0.048

[0500] 55 °C SFRS9 31.431 0.148 50 °C HPRT 30.943 0.145 55 °C HPRT 31.538 0.073

[0501] 50 °C SFRS9 29.689 0.017 55 °C SFRS9 31.449 0.022 50 °C HPRT 32.270 0.125 55 °C HPRT 33.306 0.219 F156V

[0502] 50 °C SFRS9 31.279 0.289 55 °C SFRS9 33.544 0.149 50 °C HPRT 30.507 0.124 G138S 55 °C HPRT 31.388 0.06150 °C SFRS9 29.535 0.138 55 °C SFRS9 31.167 0.174 50 °C HPRT 30.435 0.084 55 °C HPRT 31.640 0.042 C1424S

[0503] 50 °C SFRS9 29.593 0.116 55 °C SFRS9 31.151 0.131 50 °C HPRT 31.016 0.142 55 °C HPRT 31.174 0.221

[0504]

[0505] G504A 50 °C SFRS9 29.998 0.081

[0506] 55 °C SFRS9 30.712 0.106 50 °C HPRT 31.112 0.069 55 °C HPRT 32.074 0.257

[0507]

[0508] H503R 50 °C SFRS9 30.146 0.197

[0509] 55 °C SFRS9 31.517 0.155 50 °C HPRT 30.686 0.209 55 °C HPRT 31.541 0.083 I617V 50 °C SFRS9 29.447 0.029

[0510] 55 °C SFRS9 31.257 0.244 50 °C HPRT 30.441 0.098 55 °C HPRT 31.475 0.220 K550W

[0511] 50 °C SFRS9 29.624 0.021 55 °C SFRS9 31.078 0.078 50 °C HPRT 32.445 0.206 55 °C HPRT 33.547 0.120

[0512]

[0513] L136A 50 °C SFRS9 31.468 0.071

[0514] 55 °C SFRS9 33.390 0.018 50 °C HPRT 30.535 0.248 55 °C HPRT 31.196 0.141

[0515]

[0516] 50 °C SFRS9 29.538 0.008 55 °C SFRS9 30.830 0.055 50 °C HPRT 31.233 0.095 55 °C HPRT 31.978 0.082

[0517]

[0518] L491T 50 °C SFRS9 30.344 0.054

[0519] 55 °C SFRS9 32.040 0.179 50 °C HPRT 31.442 0.107 55 °C HPRT 31.910 0.191

[0520]

[0521] L528V 50 °C SFRS9 30.550 0.093

[0522] 55 °C SFRS9 31.477 0.048 50 °C HPRT 30.632 0.087 55 °C HPRT 31.827 0.106

[0523]

[0524] 50 °C SFRS9 29.401 0.052 55 °C SFRS9 31.366 0.111 50 °C HPRT 30.604 0.007 55 °C HPRT 31.806 0.299

[0525]

[0526] 50 °C SFRS9 29.617 0.084 55 °C SFRS9 31.271 0.13550 °C HPRT 40.000 0.000 MMLV-II 55 °C HPRT 40.000 0.000

[0527] V’ 50 °C SFRS9 36.936 0.850

[0528] 55 °C SFRS9 39.315 0.889 50 °C HPRT 30.734 0.059. 55 °C HPRT 31.402 0.040

[0529]

[0530] M63 Variant 50 °C SFRS9 29.675 0.038

[0531] 55 °C SFRS9 31.237 0.044 50 °C HPRT 30.246 0.171 55 °C HPRT 30.746 0.042 V433T 50 °C SFRS9 29.188 0.100

[0532] 55 °C SFRS9 30.328 0.134 50 °C HPRT 31.709 0.156 P196A 55 °C HPRT 32.805 0.158

[0533] 50 °C SFRS9 31.200 0.117 55 °C SFRS9 33.013 0.243 50 °C HPRT 31.775 0.315 P196H 55 °C HPRT 33.340 0.153

[0534] 50 °C SFRS9 31.576 0.055 55 °C SFRS9 33.208 0.198 50 °C HPRT 30.780 0.097 55 °C HPRT 32.401 0.227

[0535]

[0536] 50 °C SFRS9 30.413 0.047 55 °C SFRS9 32.569 0.081 50 °C HPRT 32.747 0.152 55 °C HPRT 33.794 0.501

[0537]

[0538] U50 °C SFRS9 31.935 0.050

[0539] 55 °C SFRS9 33.788 0.320 50 °C HPRT 31.317 0.064 55 °C HPRT 32.628 0.172

[0540]

[0541] Q374L 50 °C SFRS9 30.416 0.116

[0542] 55 °C SFRS9 32.123 0.232 50 °C HPRT 33.361 0.270 55 °C HPRT 35.522 0.721 Q530V 50 °C SFRS9 32.713 0.107

[0543] 55 °C SFRS9 34.506 0.246 50 °C HPRT 29.998 0.158 55 °C HPRT 31.010 0.166

[0544]

[0545] 50 °C SFRS9 29.448 0.044 55 °C SFRS9 30.999 0.131 50 °C HPRT 30.298 0.113 R9A 55 °C HPRT 31.237 0.112

[0546] 50 °C SFRS9 29.285 0.035 55 °C SFRS9 30.754 0.110 50 °C HPRT 31.199 0.161 R9L 55 °C HPRT 31.820 0.20450 °C SFRS9 30.188 0.077 55 °C SFRS9 31.907 0.176 50 °C HPRT 31.641 0.023 55 °C HPRT 32.731 0.219 S137F 50 °C SFRS9 30.676 0.107

[0547] 55 °C SFRS9 32.405 0.024 50 °C HPRT 31.752 0.175 55 °C HPRT 32.274 0.233

[0548]

[0549] T427C 50 °C SFRS9 30.586 0.041

[0550] 55 °C SFRS9 32.395 0.149 50 °C HPRT 30.225 0.021 55 °C HPRT 31.120 0.197

[0551]

[0552] T467N 50 °C SFRS9 29.362 0.089

[0553] 55 °C SFRS9 30.955 0.198 50 °C HPRT 31.255 0.091 55 °C HPRT 32.112 0.178 T505S 50 °C SFRS9 30.371 0.080

[0554] 55 °C SFRS9 31.948 0.020 50 °C HPRT 31.615 0.035 55 °C HPRT 32.819 0.278

[0555]

[0556] V129Y 50 °C SFRS9 30.470 0.265

[0557] 55 °C SFRS9 32.502 0.112 50 °C HPRT 35.407 0.394 55 °C HPRT 38.654 1.904 V370S 50 °C SFRS9 34.462 0.114

[0558] 55 °C SFRS9 37.876 1.931 50 °C HPRT 32.767 0.134 55 °C HPRT 33.886 0.136 V380S 50 °C SFRS9 32.219 0.086

[0559] 55 °C SFRS9 33.767 0.184 50 °C HPRT 31.325 0.061 55 °C HPRT 32.540 0.331

[0560]

[0561] Y376I 50 °C SFRS9 30.340 0.051

[0562] 55 °C SFRS9 32.130 0.109

[0563] TABLE 13

[0564] Two-Step cDNA synthesis by single mutants of modified MMEV RTase. Data was generated via qPCR assay for HPRT OR SFRS9, as indicated by the “Target Name” column. Data is reported as Ct values. K jr v nT • cDNA Target Ct Ct MMLV RTase Variant

[0565] Temp Name Mean SD 60 °C HPRT 31.731 0.278 65 °C HPRT 32.519 0.305

[0566]

[0567] A436S 60 °C SFRS9 32.960 0.132

[0568] 65 °C SFRS9 33.279 0.191 A502STOP 60 °C HPRT 33.361 0.55165 °C HPRT 33.984 0.176 60 °C SFRS9 32.748 0.374 65 °C SFRS9 33.040 0.122 60 °C HPRT 31.984 0.019 65 °C HPRT 34.310 0.660

[0569]

[0570] 60 °C SFRS9 32.501 0.313 65 °C SFRS9 33.668 0.376 60 °C HPRT 32.067 0.000 65 °C HPRT 33.061 0.230 An19T

[0571] 60 °C SFRS9 33.125 0.889 65 °C SFRS9 33.301 0.279 60 °C HPRT 30.593 0.060 65 °C HPRT 30.683 0.225

[0572]

[0573] C157A 60 °C SFRS9 31.248 0.147

[0574] 65 °C SFRS9 31.767 0.216 60 °C HPRT 32.614 0.498 65 °C HPRT 33.940 0.395

[0575]

[0576] 60 °C SFRS9 32.788 0.082 65 °C SFRS9 33.710 0.239 60 °C HPRT 32.236 0.062 65 °C HPRT 34.047 0.683

[0577]

[0578] 60 °C SFRS9 32.992 0.138 65 °C SFRS9 33.775 0.650 60 °C HPRT 31.463 0.365 65 °C HPRT 32.386 0.215

[0579]

[0580] 60 °C SFRS9 32.193 0.217 65 °C SFRS9 32.756 0.222 60 °C HPRT 31.393 0.105 65 °C HPRT 32.349 0.719 E607S 60 °C SFRS9 32.384 0.120

[0581] 65 °C SFRS9 32.993 0.344 60 °C HPRT 31.617 0.163 65 °C HPRT 32.247 0.266 E7P 60 °C SFRS9 32.172 0.203

[0582] 65 °C SFRS9 33.015 0.283 60 °C HPRT 33.830 0.038 65 °C HPRT 36.150 2.737 F156V

[0583] 60 °C SFRS9 35.000 0.485 65 °C SFRS9 37.584 1.810 60 °C HPRT 31.423 0.196 65 °C HPRT 32.009 0.453

[0584]

[0585] G138S 60 °C SFRS9 32.225 0.005

[0586] 65 °C SFRS9 32.871 0.177 60 °C HPRT 31.021 0.451 G424S 65 °C HPRT 33.381 0.823

[0587] 60 °C SFRS9 32.342 0.15865 °C SFRS9 33.948 0.617 60 °C HPRT 32.078 0.632 65 °C HPRT 33.188 0.577

[0588]

[0589] U5U60 °C SFRS9 32.544 0.185

[0590] 65 °C SFRS9 34.508 0.555 60 °C HPRT 31.671 0.431 65 °C HPRT 32.834 0.541

[0591]

[0592] 5UJ60 °C SFRS9 32.847 0.109

[0593] 65 °C SFRS9 33.373 0.178 60 °C HPRT 30.928 0.383 65 °C HPRT 32.212 0.6666 7V60 °C SFRS9 31.790 0.048

[0594] 65 °C SFRS9 33.312 0.269 60 °C HPRT 30.983 0.303 65 °C HPRT 32.524 0.270 K550W

[0595] 60 °C SFRS9 32.140 0.161 65 °C SFRS9 32.873 0.064 60 °C HPRT 33.733 0.289 65 °C HPRT 34.707 0.663

[0596]

[0597] 60 °C SFRS9 34.960 0.555 65 °C SFRS9 35.916 1.108 60 °C HPRT 31.601 0.541 65 °C HPRT 32.513 0.196

[0598]

[0599] 60 °C SFRS9 32.134 0.176 65 °C SFRS9 33.464 0.261 60 °C HPRT 31.859 0.455 65 °C HPRT 32.907 0.777

[0600]

[0601] 960 °C SFRS9 33.131 0.214

[0602] 65 °C SFRS9 33.761 0.466 60 °C HPRT 31.544 0.022 65 °C HPRT 32.679 0.120

[0603]

[0604] 5 8V60 °C SFRS9 32.599 0.192

[0605] 65 °C SFRS9 33.699 0.271 60 °C HPRT 30.756 0.088 65 °C HPRT 31.846 0.651

[0606]

[0607] 5 860 °C SFRS9 31.695 0.042

[0608] 65 °C SFRS9 32.307 0.173 60 °C HPRT 32.995 0.493 65 °C HPRT 35.138 0.956

[0609]

[0610] 6U° 60 °C SFRS9 32.745 0.136

[0611] 65 °C SFRS9 33.955 0.141 60 °C HPRT 40.000 0.000 MM V TT65°C HPRT 40-000°-000

[0612] V’ 60 °C SFRS9 37.581 0.870

[0613] 65 °C SFRS9 36.984 0.422 M63 Variant 60 °C HPRT 30.858 0.18965 °C HPRT 32.182 0.289 60 °C SFRS9 31.793 0.075 65 °C SFRS9 33.278 0.193 60 °C HPRT 30.326 0.067 65 °C HPRT 31.855 0.353

[0614]

[0615] V 5560 °C SFRS9 31.561 0.114

[0616] 65 °C SFRS9 32.620 0.164 60 °C HPRT 34.207 0.193 pnii rQ AA65 °C HPRT 36.194 0.209

[0617] 60 °C SFRS9 34.614 0.223 65 °C SFRS9 35.902 1.173 60 °C HPRT 35.810 0.459 65 °C HPRT 36.612 2.407 r 196H

[0618] 60 °C SFRS9 35.249 0.421 65 °C SFRS9 35.989 0.498 60 °C HPRT 32.766 0.426 65 °C HPRT 34.145 1.717

[0619]

[0620] 60 °C SFRS9 33.498 0.256 65 °C SFRS9 34.127 0.548 60 °C HPRT 32.972 0.486 65 °C HPRT 33.634 0.575

[0621]

[0622] 60 °C SFRS9 34.478 0.504 65 °C SFRS9 34.537 0.191 60 °C HPRT 32.656 0.615 65 °C HPRT 33.415 0.546

[0623]

[0624] A500Q 60 °C SFRS9 33.467 0.290

[0625] 65 °C SFRS9 34.399 0.343 60 °C HPRT 33.815 0.425 65 °C HPRT 34.956 0.62453UV60 °C SFRS9 35.200 0.555

[0626] 65 °C SFRS9 36.259 0.421 60 °C HPRT 31.211 0.290 65 °C HPRT 31.513 0.189

[0627]

[0628] 7360 °C SFRS9 32.178 0.064

[0629] 65 °C SFRS9 32.481 0.068 60 °C HPRT 30.775 0.052rArA A65 °C HPRT 31.782 0.401

[0630] 60 °C SFRS9 31.537 0.101 65 °C SFRS9 32.478 0.023 60 °C HPRT 31.648 0.306 65 °C HPRT 34.292 1.344

[0631]

[0632] 960 °C SFRS9 32.768 0.311

[0633] 65 °C SFRS9 33.965 0.260 60 °C HPRT 33.357 0.163 S137F 65 °C HPRT 35.658 0.675

[0634] 60 °C SFRS9 33.571 0.06965 °C SFRS9 35.028 0.370

[0635] 60 °C HPRT 32.056 0.313

[0636] 65 °C HPRT 34.923 0.979

[0637]

[0638] 7L60 °C SFRS9 33.533 0.078

[0639] 65 °C SFRS9 34.649 0.284

[0640] 60 °C HPRT 31.989 0.356

[0641] 65 °C HPRT 33.114 0.301 1467N

[0642] 60 °C SFRS9 32.624 0.124

[0643] 65 °C SFRS9 33.135 0.207

[0644] 60 °C HPRT 32.766 0.217

[0645] 65 °C HPRT 33.316 0.3455U5S60 °C SFRS9 32.922 0.195

[0646] 65 °C SFRS9 33.424 0.267

[0647] 60 °C HPRT 32.998 0.559

[0648] 65 °C HPRT 34.176 0.163

[0649]

[0650] 60 °C SFRS9 33.911 0.140

[0651] 65 °C SFRS9 34.535 0.185

[0652] 60 °C HPRT 35.659 0.779

[0653] 65 °C HPRT 37.014 2.166

[0654]

[0655] 60 °C SFRS9 38.050 0.842

[0656] 65 °C SFRS9 36.458 0.411

[0657] 60 °C HPRT 33.963 1.162

[0658] 65 °C HPRT 35.138 0.360

[0659]

[0660] 60 °C SFRS9 35.229 0.453

[0661] 65 °C SFRS9 34.984 0.171

[0662] 60 °C HPRT 33.342 0.904

[0663] 65 °C HPRT 34.003 0.701

[0664]

[0665] 37660 °C SFRS9 32.997 0.164

[0666] 65 °C SFRS9 33.912 0.129

[0667] Example 6 — Evaluation of additional MMLV Reverse Transcriptase Single Mutants.

[0668] A further set of single mutants of the M63 variant base construct were generated to examine their activity and thermostability. In particular, A477Y, A587T, G524N, P474S, T523L, and W593L variants were generated by site directed mutagenesis according to the method described subsequently with respect to Example 1. All mutations were made to the M63 variant base sequence. The amino acid sequences of the generated mutants are provided in Table 14, below.

[0669] _ TABLE 14 _

[0670] MMLV RTase

[0671] Variant Name SEQ ID _ Amino Acid Sequence (N - C) _M63 + A477Y 294 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDT DRVQFGPVVYENPATEEPEPEEGEQHNCEDIEAEAHGT RPDETDQPEPDADHTWYTGGSSEEQEGQRKAGAAVTT ETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKEN VYTNSRYAFATAHWHGEIYRRRGEETSEGKEIKNKDEI EAEEKAEFEPKRESIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTEEIENSSPYTSEHF

[0672] M63 + A587T 295 MGSSHHHHHHSSGEVPRGSHMTENIEDEHREHETSKEP DVSEGSTWESDFPQAWAETGGMGEAVRQAPEIIPEKAT STPVSIKQYPMSREAREGIKPHIRREYDQGIEVPCQSPW NTPERPVKKPGTNDYRPVQDEREVNKRVEDIHPTVPNP YNEESGEPPSHQWYTVEDEKDAFFCEREHPTSQPEFAF EWRDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDT DRVQFGPVVAENPATEEPEPEEGEQHNCEDIEAEAHGT RPDETDQPEPDADHTWYTGGSSEEQEGQRKAGAAVTT ETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKEN VYTNSRYTFATAHWHGEIYRRRGEETSEGKEIKNKDEI EAEEKAEFEPKRESIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTEEIENSSPYTSEHF

[0673] M63 + G524N 296 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPYTSEHF

[0674] M63 + P474S 297 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDT DRVQFGSVVAENPATEEPEPEEGEQHNCEDIEAEAHGT RPDETDQPEPDADHTWYTGGSSEEQEGQRKAGAAVTT ETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKEN VYTNSRYAFATAHWHGEIYRRRGEETSEGKEIKNKDEI EAEEKAEFEPKRESIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTEEIENSSPYTSEHF

[0675] M63 + T523L 298 MGSSHHHHHHSSGEVPRGSHMTENIEDEHREHETSKEP DVSEGSTWESDFPQAWAETGGMGEAVRQAPEIIPEKAT STPVSIKQYPMSREAREGIKPHIRREYDQGIEVPCQSPW NTPERPVKKPGTNDYRPVQDEREVNKRVEDIHPTVPNP YNEESGEPPSHQWYTVEDEKDAFFCEREHPTSQPEFAF EWRDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDT DRVQFGPVVAENPATEEPEPEEGEQHNCEDIEAEAHGT RPDETDQPEPDADHTWYEGGSSEEQEGQRKAGAAVTT ETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKEN VYTNSRYAFATAHWHGEIYRRRGEETSEGKEIKNKDEI EAEEKAEFEPKRESIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTEEIENSSPYTSEHF

[0676] M63 + W593L 299 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHLHGEIYRRRGLLTSEGKEIKNKDEIL ALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPYTSEHF

[0677] The ability of each combination mutant to synthesize cDNA from purified total Universal RNA (Agilent) was compared to purified protein having the sequence of SSII (Invitrogen; SEQ ID NO: 276) and the M63 variant base construct. In particular, the two-step assay described in the discussion of Example 3 was used with target temperatures of 50 °C, 55 °C, 60 °C, 65 °C and a final concentration of reverse transcriptase of 5.2 nM. The two-step reactions for the MMLV variants were analyzed and reported by Ct value. Table 12 reports Ct values for each variant (including the M63 and MMLV-II variants) at 50 °C and 55 °C, and Table 13 Ct values for each variant (including the M63 and MMLV-II variants) at 60 °C and 65 °C.

[0678] As is shown in the data of Table 15, all examined mutants showed significantly improved activity as compared to the MMLV-II variant at all examined temperatures. Further, all mutants showed comparable activity as compared to the M63 variant MMLV RTase.

[0679] TABLE 15

[0680] Two-Step cDNA synthesis by triple and quadruple mutants of modified MMLV RTase. Data was generated via qPCR assay for HPRT OR SFRS9, as indicated by the “Target Name” column. Data is reported as

[0681] Ct values.

[0682] Kojr v nT w • cDNA Target Ct Ct

[0683]

[0684] Temp Name Mean SD

[0685] 50 °C HPRT 32.487 0.231

[0686] 55 °C HPRT 33.616 0.104

[0687] 60 °C HPRT 34.897 0.277

[0688] . 65 °C HPRT 35.872 0.441 A477Y

[0689] 50 °C SFRS9 31.958 0.149

[0690] 55 °C SFRS9 33.505 0.170

[0691] 60 °C SFRS9 34.283 0.229

[0692] 65 °C SFRS9 35.025 0.226

[0693] 50 °C HPRT 31.129 0.238

[0694] 55 °C HPRT 31.810 0.055

[0695] 60 °C HPRT 33.289 0.184

[0696] 65 °C HPRT 34.014 0.336

[0697]

[0698] 50 °C SFRS9 30.249 0.080

[0699] 55 °C SFRS9 31.659 0.093

[0700] 60 °C SFRS9 32.636 0.022

[0701] 65 °C SFRS9 33.224 0.126

[0702] 50 °C HPRT 31.275 0.166 ^n / 1XT55 °C HPRT 32.201 0.104 G524N

[0703] 60 °C HPRT 32.845 0.064

[0704] 65 °C HPRT 33.619 0.16450 °C SFRS9 30.344 0.035 55 °C SFRS9 31.711 0.121 60 °C SFRS9 32.664 0.171 65 °C SFRS9 33.201 0.037 50 °C HPRT 31.079 0.021 55 °C HPRT 31.521 0.136 60 °C HPRT 32.599 0.190. 65 °C HPRT 33.020 0.257

[0705]

[0706] 63 Variant50oCSFRS9 30.022 0.108

[0707] 55 °C SFRS9 31.255 0.192 60 °C SFRS9 32.043 0.156 65 °C SFRS9 32.701 0.061 50 °C HPRT 31.997 0.026 55 °C HPRT 32.704 0.091 60 °C HPRT 33.897 0.272 65 °C HPRT 34.074 0.086 P474S

[0708] 50 °C SFRS9 31.491 0.045 55 °C SFRS9 32.824 0.081 60 °C SFRS9 33.147 0.082 65 °C SFRS9 33.863 0.176 50 °C HPRT 31.019 0.108 55 °C HPRT 31.633 0.115 60 °C HPRT 32.845 0.195 65 °C HPRT 33.481 0.2355 350 °C SFRS9 30.035 0.050

[0709] 55 °C SFRS9 31.219 0.133 60 °C SFRS9 32.222 0.142 65 °C SFRS9 32.955 0.028 50 °C HPRT 30.896 0.121 55 °C HPRT 31.990 0.086 60 °C HPRT 34.203 0.333 65 °C HPRT 34.147 0.272

[0710]

[0711] 50 °C SFRS9 30.234 0.052 55 °C SFRS9 32.140 0.085 60 °C SFRS9 33.323 0.198 65 °C SFRS9 34.002 0.228 50 °C HPRT 40.000 0.000 55 °C HPRT 40.000 0.000 60 °C HPRT 40.000 0.000 MM1 V TT65°C HPRT 39 952 0 068

[0712] V’ 50 °C SFRS9 37.108 0.273

[0713] 55 °C SFRS9 38.016 0.183 60 °C SFRS9 39.708 0.299 65 °C SFRS9 38.310 0.253Example 6 — Evaluation of additional MMLV Reverse Transcriptase Single Mutants and further combination mutants.

[0714] A further set of single mutants of the M63 variant base construct were generated as were a set of combination mutants of advantageous mutants identified Example 3 and Example 5. In particular, A477Y, A587T, G524N, P474S, T523L, and W593L variants were generated by site directed mutagenesis according to the method described subsequently with respect to Example 1. All mutations were made to the M63 variant base sequence. The amino acid sequences of the generated mutants are provided in Table 16, below.

[0715] _ TABLE 16 _

[0716] MMLV RTase

[0717] Variant Name SEQ ID _ Amino Acid Sequence (N - C) _

[0718] M63 + A500Q 340 MGSSHHHHHHSSGEVPRGSHMTENIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQETWTRLPQGFKNSPTLFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TLGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTLFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKLGPWRRPVAY ESKKLDPVAAGWPPCERMVAAIAVETKDAGKLTMGQ PENIEAPHAVEAEVKQPPDRWLSNARMTHYQAEEEDT DRVQFGPVVAENPATLEPEPEEGEQHNCEDIEQEAHGT RPDETDQPEPDADHTWYTGGSSEEQEGQRKAGAAVTT ETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGEETSEGKEIKNKDEI EAEEKAEFEPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLEIENSSPYTSEHF

[0719] M63 + A619R 341 MGSSHHHHHHSSGEVPRGSHMTENIEDEHREHETSKEP DVSEGSTWLSDFPQAWAETGGMGEAVRQAPEIIPEKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGIEVPCQSPW NTPERPVKKPGTNDYRPVQDEREVNKRVEDIHPTVPNP YNEESGEPPSHQWYTVEDEKDAFFCERLHPTSQPEFAF EWRDPEMGISGQETWTRLPQGFKNSPTLFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TLGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTLFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKLGPWRRPVAY ESKKLDPVAAGWPPCERMVAAIAVETKDAGKLTMGQ PENIEAPHAVEAEVKQPPDRWLSNARMTHYQAEEEDT DRVQFGPVVAENPATLEPEPEEGEQHNCEDIEAEAHGT RPDETDQPEPDADHTWYTGGSSEEQEGQRKAGAAVTT ETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGEETSEGKEIKNKDEI EREEKAEFEPKRESIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPYTSEHF

[0720] M63 + R629T 342 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDT DRVQFGPVVAENPATEEPEPEEGEQHNCEDIEAEAHGT RPDETDQPEPDADHTWYTGGSSEEQEGQRKAGAAVTT ETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKEN VYTNSRYAFATAHWHGEIYRRRGEETSEGKEIKNKDEI EAEEKAEFEPKTESIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0721] M63 + 343 MGSSHHHHHHSSGEVPRGSHMTENIEDEHAEHETSKEP R9A / C157A DVSEGSTWESDFPQAWAETGGMGEAVRQAPEIIPEKAT STPVSIKQYPMSREAREGIKPHIRREYDQGIEVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNEESGEPPSHQWYTVEDEKDAFFAEREHPTSQPEFAF EWRDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDT DRVQFGPVVAENPATEEPEPEEGEQHNCEDIEAEAHGT RPDETDQPEPDADHTWYTGGSSEEQEGQRKAGAAVTT ETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKEN VYTNSRYAFATAHWHGEIYRRRGEETSEGKEIKNKDEI EAEEKAEFEPKRESIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTEEIENSSPYTSEHF

[0722] M63 + 344 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP C157A / R159P DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFALPLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0723] M63 + 345 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP C157A / R159S DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFALSLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0724] M63 + 346 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP C157A / R173E DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFALRLHPTSQPLFAF EWEDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLA DFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDA RKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMA APLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALG LPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTR PDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNV YTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEIL ALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0725] M63 + 347 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP C157A / T287D DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFALRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0726] M63 + 348 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP C157A / L333E DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFALRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0727] M63 + 349 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP C157A / V433T DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFALRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLTILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0728] M63 + 350 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP C157A / K435H DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFALRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNIHAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0729] M63 + 351 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP C157A / T467N DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFALRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDN DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0730] M63 + 352 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP C157A / G490A DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFALRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEALQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0731] M63 + 353 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP C157A / G490A DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFALRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEALQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEIEAEEKAEFEPKRESIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTEEIENSSPYTSEHF

[0732] M63 + 354 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP C157A / A502S DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT TOPSTPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFALRLHPTSQPLFAF EWRDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDT DRVQFGPVVAENPATEEPEPEEGEQHNCEDIEAE

[0733] M63 + 355 MGSSHHHHHHSSGEVPRGSHMTENIEDEHREHETSKEP C157A / L528Y DVSEGSTWESDFPQAWAETGGMGEAVRQAPEIIPEKAT STPVSIKQYPMSREAREGIKPHIRREYDQGIEVPCQSPW NTPERPVKKPGTNDYRPVQDEREVNKRVEDIHPTVPNP YNEESGEPPSHQWYTVEDEKDAFFAEREHPTSQPEFAF EWRDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDT DRVQFGPVVAENPATEEPEPEEGEQHNCEDIEAEAHGT RPDETDQPEPDADHTWYTGGSSYEQEGQRKAGAAVTT ETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKEN VYTNSRYAFATAHWHGEIYRRRGEETSEGKEIKNKDEI EAEEKAEFEPKRESIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTEEIENSSPYTSEHF

[0734] M63 + 356 MGSSHHHHHHSSGEVPRGSHMTENIEDEHREHETSKEP C157A / R629M DVSEGSTWESDFPQAWAETGGMGEAVRQAPEIIPEKAT STPVSIKQYPMSREAREGIKPHIRREYDQGIEVPCQSPW NTPERPVKKPGTNDYRPVQDEREVNKRVEDIHPTVPNP YNEESGEPPSHQWYTVEDEKDAFFAEREHPTSQPEFAF EWRDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDT DRVQFGPVVAENPATEEPEPEEGEQHNCEDIEAEAHGT RPDETDQPEPDADHTWYTGGSSEEQEGQRKAGAAVTT ETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKEN VYTNSRYAFATAHWHGEIYRRRGEETSEGKEIKNKDEI EAEEKAEFEPKMESIIHCPGHQKGHSAEARGNRMADQ AARKAAITETPDTSTEEIENSSPYTSEHF

[0735] M63 + 357 MGSSHHHHHHSSGEVPRGSHMTENIEDEHREHETSKEP C157A / A646V DVSEGSTWESDFPQAWAETGGMGEAVRQAPEIIPEKATSTPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFALRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEVRGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0736] M63 + 358 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHALHETSKEP R9A / R159P DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLPLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0737] M63 + 359 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHALHETSKEP R9A / R159S DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLSLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0738] M63 + 360 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHALHETSKEP R9A / R173E DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWEDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDEA DFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDA RKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMA APLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALG LPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTR PDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNV YTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEIL ALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0739] M63 + 361 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHALHETSKEP R9A / T287D DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0740] M63 + 362 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHALHETSKEP R9A / L333E DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0741] M63 + 363 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHALHETSKEP R9A / V433T DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLTILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0742] M63 + 364 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHALHETSKEP R9A / K435H DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNIHAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0743] M63 + 365 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHALHETSKEP R9A / T467N DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDN DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0744] M63 + 366 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHALHETSKEP R9A / G490A DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEALQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0745] M63 + 367 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHALHETSKEP R9A / A502STO DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT P STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAE

[0746] M63 + 368 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHALHETSKEP R9A / L528Y DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSYLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0747] M63 + 369 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHALHETSKEP R9A / R629M DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKMLSIIHCPGHQKGHSAEARGNRMADQ AARKAAITETPDTSTLLIENSSPYTSEHF

[0748] M63 + 370 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHALHETSKEP R9A / A646V DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEVRGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0749] M63 + 371 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R159P / R173E DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLPLHPTSQPLFAF EWEDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLA DFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDA RKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMA APLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALG LPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTR PDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNV YTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEIL ALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0750] M63 + 372 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R159P / V433T DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLPLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLTILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0751] M63 + 373 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R159P / T467N DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLPLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDN DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0752] M63 + 374 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R159P / A502ST DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT OP STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLPLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAE

[0753] M63 + 375 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R159P / L528Y DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLPLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSYLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0754] M63 + 376 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R159S / R173E DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLSLHPTSQPLFAF EWEDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLA DFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDA RKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMA APLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALG LPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTR PDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNV YTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEIL ALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0755] M63 + 377 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R159S / V433T DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLSLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLTILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0756] M63 + 378 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R159S / T467N DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLSLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDN DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0757] M63 + 379 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R159S / A502ST DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT OP STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLSLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAE

[0758] M63 + 380 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R159S / L528Y DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLSLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSYLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0759] M63 + 381 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R173E / T287D DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWEDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLA DFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDA RKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0760] M63 + 382 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R173E / L333E DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWEDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLA DFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDA RKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMA APLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPALG LPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTR PDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNV YTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEIL ALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0761] M63 + 383 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R173E / V433T DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWEDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLA DFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDA RKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMA APLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALG LPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LTILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTR PDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNV YTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEIL ALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0762] M63 + 384 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R173E / K435H DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWEDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLA DFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDA RKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMA APLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALG LPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LNIHAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTR PDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNV YTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0763] M63 + 385 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R173E / T467N DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWEDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDNDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPYTSEHF

[0764] M63 + 386 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEPR173E / G490A DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPWNTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWEDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEALQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPYTSEHF

[0765] M63 + 387 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R173E / A502ST DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT OP STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWEDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLA DFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDA RKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMA APLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALG LPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAE

[0766] M63 + 388 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R173E / L528Y DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWEDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGGSSYLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPYTSEHF

[0767] M63 + 389 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R173E / R629M DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWEDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLA DFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDA RKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMA APLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALG LPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTR PDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNV YTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEIL ALLKALFLPKMLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0768] M63 + 390 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R173E / A646V DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWEDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLA DFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDA RKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEMA APLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALG LPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTR PDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNV YTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEIL ALLKALFLPKRLSIIHCPGHQKGHSAEVRGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0769] M63 + 391 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / V433T DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLTILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0770] M63 + 392 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / T467N DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD NDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0771] M63 + 393 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / A502ST DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT OP STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAE

[0772] M63 + 394 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / L528Y DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSYLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0773] M63 + 395 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP L333E / V433T DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLTILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0774] M63 + 396 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP L333E / T467N DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0775] M63 + 397 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP L333E / A502ST DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT OP STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAE

[0776] M63 + 398 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP L333E / L528Y DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSYLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0777] M63 + 399 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP V433T / K435H DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLTIHAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0778] M63 + 400 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP V433T / T467N DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLTILAPHAVEALVKQPPDRWLSNARMTHYQALLLDN DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0779] M63 + 401 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP V433T / G490A DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLTILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEALQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0780] M63 + 402 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP V433T / A502ST DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT OP STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLTILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAE

[0781] M63 + 403 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP V433T / L528Y DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLTILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSYLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0782] M63 + 404 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP V433T / R629M DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLTILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKMLSIIHCPGHQKGHSAEARGNRMADQ AARKAAITETPDTSTLLIENSSPYTSEHF

[0783] M63 + 405 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP V433T / A646V DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLTILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEVRGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0784] M63 + 406 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP K435H / T467N DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNIHAPHAVEALVKQPPDRWLSNARMTHYQALLLDN DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0785] M63 + 407 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP K435H / A502S DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT TOPSTPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNIHAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAE

[0786] M63 + 408 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP K435H / L528Y DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNIHAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSYLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0787] M63 + 409 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T467N / G490A DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDN DRVQFGPVVALNPATLLPLPEEALQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHFM63 + 410 MGSSHHHHHHSSGEVPRGSHMTENIEDEHREHETSKEP T467N / A502ST DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT OP STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDN DRVQFGPVVAENPATEEPEPEEGEQHNCEDIEAE

[0788] M63 + 411 MGSSHHHHHHSSGEVPRGSHMTENIEDEHREHETSKEP T467N / L528Y DVSEGSTWESDFPQAWAETGGMGEAVRQAPEIIPEKAT STPVSIKQYPMSREAREGIKPHIRREYDQGIEVPCQSPW NTPERPVKKPGTNDYRPVQDEREVNKRVEDIHPTVPNP YNEESGEPPSHQWYTVEDEKDAFFCEREHPTSQPEFAF EWRDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDN DRVQFGPVVAENPATEEPEPEEGEQHNCEDIEAEAHGT RPDETDQPEPDADHTWYTGGSSYEQEGQRKAGAAVTT ETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKEN VYTNSRYAFATAHWHGEIYRRRGEETSEGKEIKNKDEI EAEEKAEFEPKRESIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTEEIENSSPYTSEHF

[0789] M63 + 412 MGSSHHHHHHSSGEVPRGSHMTENIEDEHREHETSKEP T467N / R629M DVSEGSTWESDFPQAWAETGGMGEAVRQAPEIIPEKAT STPVSIKQYPMSREAREGIKPHIRREYDQGIEVPCQSPW NTPERPVKKPGTNDYRPVQDEREVNKRVEDIHPTVPNP YNEESGEPPSHQWYTVEDEKDAFFCEREHPTSQPEFAF EWRDPEMGISGQETWTREPQGFKNSPTEFDEAEHRDE ADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQ TEGNEGYRASAKKAQICQKQVKYEGYEEKEGQRWITD ARKETVMGQPTPKTPREEREFEGKAGFCREWIPGFAEM AAPEYPETKTGTEFNWGPDQQKAYQEIKQAEETAPAE GEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPVAY ESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PENIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDN DRVQFGPVVAENPATEEPEPEEGEQHNCEDIEAEAHGT RPDETDQPEPDADHTWYTGGSSEEQEGQRKAGAAVTT ETEVIWAKAEPAGTSAQRAQEIAETQAEKMAEGKKEN VYTNSRYAFATAHWHGEIYRRRGEETSEGKEIKNKDEI EAEEKAEFEPKMESIIHCPGHQKGHSAEARGNRMADQ AARKAAITETPDTSTEEIENSSPYTSEHF

[0790] M63 + 413 MGSSHHHHHHSSGEVPRGSHMTENIEDEHREHETSKEP T467N / A646V DVSEGSTWESDFPQAWAETGGMGEAVRQAPEIIPEKAT STPVSIKQYPMSREAREGIKPHIRREYDQGIEVPCQSPW NTPERPVKKPGTNDYRPVQDEREVNKRVEDIHPTVPNP YNEESGEPPSHQWYTVEDEKDAFFCEREHPTSQPEFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDN DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEVRGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0791] M63 + 414 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP G490A / A502S DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT TOPSTPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEALQHNCLDILAE

[0792] M63 + 415 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP G490A / L528Y DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEALQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSYLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0793] M63 + 416 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP L528Y / R629M DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSYLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKMLSIIHCPGHQKGHSAEARGNRMADQ AARKAAITETPDTSTLLIENSSPYTSEHF

[0794] M63 + 417 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP L528Y / A646V DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKETVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSYLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEVRGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0795] M63 + 418 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHALHETSKEP R9A / T287D / L3 DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT 33E STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0796] M63 + 419 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP C157A / T287D / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT L333E STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFALRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0797] M63 + 420 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP R173E / T287D / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT L333E STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWEDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLA DFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDA RKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAEM AAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGT RPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLN VYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSPYTSEHF

[0798] M63 + 421 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / L333E / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT V433T STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLTILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0799] M63 + 422 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / L333E / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT T467N STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD NDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0800] M63 + 423 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / L333E / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT A502STOPSTPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAE

[0801] M63 + 424 MGSSHHHHHHSSGLVPRGSHMTLNIEDEHRLHETSKEP T287D / L333E / DVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT L528Y STPVSIKQYPMSREARLGIKPHIRRLYDQGILVPCQSPW NTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITD ARKEDVMGQPTPKTPRELREFLGKAGFCRLWIPGFAE MAAPLYPLTKTGTEFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLNILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTGGSSYLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHWHGEIYRRRGLLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAD QAARKAAITETPDTSTLLIENSSPYTSEHF

[0802] The ability of each mutant to synthesize cDNA from purified total Universal RNA (Agilent) was compared to purified protein having the sequence of SSII (Invitrogen; SEQ ID NO: 276) and the M63 variant base construct. In particular, the two-step assay described in the discussion of Example 3 was used with target temperatures of 50 °C, 55 °C, 60 °C, 65 °C and a final concentration of reverse transcriptase of 5.2 nM. The two-step reactions for the MMLV variants were analyzed and reported by Ct value. Table 17, Table 18, Table 19, and Table 20 report Ct values for each variant (including the M63 and MMLV-II variants) at 50 °C, 55 °C, 60 °C, and 65 °C, respectively.As is shown by the data of Tables 17-20, all mutants showed significantly improved activity as compared to the MMLV-II variant at all examined temperatures. Further, all mutants showed comparable or improved activity as compared to the M63 variant MMLV RTase at all examined temperatures. The stacked variants having the highest activity and greatest thermostability were T287D / L333E (SEQ ID NO: 418), C157A / T287D (SEQ ID NO: 419), R9A / T287D (SEQ ID NO: 361), R9A / V433T (SEQ ID NO: 363), T287D / V433T (SEQ ID NO: 391), T287D / A502STOP (SEQ ID NO: 393), V433T / A502STOP (SEQ ID NO: 402), T287D / L333E / V433T (SEQ ID NO: 421), T287D / L333E / A502STOP (SEQ ID NO: 423), and T287D / L333E / L528Y (SEQ ID NO: 424). variants showed significantly increased activity at multiple temperatures (i.e., and improved thermostability) as compared to both M63 and MMLV-II variants.

[0803] TABLE 17

[0804] Two-Step cDNA synthesis by single, double, and triple mutants of modified MMLV RTase. Data was generated via qPCR assay for HPRT OR SFRS9, as indicated by the “Target Name” column.

[0805] ■ cDNA Target Ct Ct MMLV RTase Variant „

[0806] Temp Name Mean SD

[0807] 50 °C HPRT 31.759 0.058

[0808] A500Q 50 °C SFRS9 31.221 0.025

[0809] 50 °C HPRT 31.967 0.109 A619R

[0810] 50 °C SFRS9 31.460 0.070

[0811] 50 °C HPRT 31.654 0.145

[0812]

[0813] 50 °C HPRT 31.911 0.064 POA / C 7A 50 °C SFRS9 31.185 0.074 R9A / C157A5() oc SFRS9 31 412 0 ()46

[0814] C C115577AA / / RR115599PP550()°oCc SHFPRRST93321’9U233°0-°13579

[0815] C157A / R159S50°C HPRT 32’767°’147

[0816] 50 °C SFRS9 32.653 0.128

[0817] 50 °C HPRT 30.766 0.066

[0818]

[0819] 50 °C SFRS9 30.524 0.074 C157A / T287D50°C HPRT 30’012°’011

[0820] 50 °C SFRS9 29.690 0.054 C157A / L333E 50 °C HPRT 30.754 0.049

[0821] 50 °C SFRS9 30.153 0.106 C157A / V433T50°C HPRT 30’535°’112

[0822] 50 °C SFRS9 30.048 0.077 C157A / K435H50°C HPRT 31’129°’103

[0823] 50 °C SFRS9 30.582 0.025 C157A / T467N 50 °C HPRT 31.348 0.10050 °C SFRS9 30.818 0.030 C157A / G490A 50 °C HPRT 30.742 0.116

[0824] 50 °C SFRS9 30.324 0.153 C C1n57 / AA / / AA550U22SMTOUPP55(0)°oCc SHFPRRST93209299542 002()0143

[0825] C157A / L528Y 50 °C HPRT 31.288 0.133

[0826] 50 °C SFRS9 30.812 0.037 50 °C HPRT 30.868 0.053 C157A / R629M3()

[0827] 50 °C HPRT 30.876 0.019C 57 7 6 6V50 °C SFRS9 30.364 0.044 R9A / R159P50°C HPRT 30’624°-°44

[0828] 50 °C SFRS9 30.202 0.117 R9A / R173F50°C HPRT 30’676 0220

[0829] 50 °C SFRS9 30.299 0.044 R9A / T287D 50 °C HPRT 30.149 0.066

[0830] 50 °C SFRS9 29.718 0.036 50 °C HPRT 30.609 0.095 50 °C SFRS9 29.971 0.032 R9A / V433T50°C HPRT 30281°’028

[0831] 50 °C SFRS9 29.528 0.042 R R99AA / / KK443355HH50°CsHpPRRST93300'6Q91990Q155

[0832] R9A / T467N 50 °C HPRT 31.267 0.045

[0833] 50 °C SFRS9 30.582 0.106 R9A / G490A50°C HPRT 30’868 O4°

[0834] 50 °C SFRS9 30.241 0.076 R R99AA / / AA550022SSTTGOPP5500° oCC SHFPRRST932092.87970° 0.07631 R9A / T 528Y50°C HPRT 30’980°34

[0835] 50 °C SFRS9 30.467 0.064 50 °C HPRT 30.913 0.1379 / 6 950 °C SFRS9 30.305 0.068

[0836] 50 °C HPRT 31.141 0.0689 7 6 6V50 °C SFRS9 30.651 0.081 R159P / R173F50°C HPRT 30’877°8359 7 7350 °C SFRS9 30.438 0.015 R159P / V433T50°C HPRT 30’044 O50

[0837] 50 °C SFRS9 29.642 0.023 R159P / T467N50”C HPRT 30'908 0 103

[0838] 50 °C SFRS9 30.330 0.028 R159P / A502STOP 50 °C HPRT 30.007 0.015

[0839] R R115599PP / / TL552288YY55Q0° oCC SHFPRRST9330Q237625°0 13252

[0840]

[0841] R159S / R173F50°C HPRT 31’630 046659S / 7350 °C SFRS9 31.497 0.021 R159S / V433T50°C HPRT 31’072 0433

[0842] 50 °C SFRS9 30.504 0.209 R159S / T467N50”C HPRT 32'079°-090

[0843] 50 °C SFRS9 31.483 0.065 R R 115599S8 / / AA5500228STTOOPP50°CsHpPRRST9330()300°Q'0Q5g48

[0844] R R115599SS / / TL552288YY5500° oCC SHFPRRST93301’380176°0’010870

[0845]

[0846] R173E / T287D 50 °C HPRT 30.457 0.146

[0847] 73 7 8750 °C SFRS9 30.075 0.116 R173F / 1333F50°C HPRT 30’522 0402

[0848] 50 °C SFRS9 29.999 0.082 R173E / V433T50°C HPRT 30’864°79

[0849] 50 °C SFRS9 30.295 0.078 R R117733FE / / KK443355HH5500° oCC SHFPRRST93300’603645 004.07362 P RI1T73E / / TT4 / I6™7N5500° oCC SHFPRRST93300-955566°0-.014141R173E / G490A50°C HPRT 31’304 0471

[0850] 50 °C SFRS9 30.909 0.145 R R117733FE / / AA550022SSTTOOPP5500°oCc SHFPRRST93300’ _78762600404000 R173F / T 528Y50°C HPRT 30’973 0432

[0851] 50 °C SFRS9 30.587 0.094 R R117733FE / / RR662299MM5500° oCC SHFPRRST93311 04571 ° 0.09135

[0852] 50 °C HPRT 30.891 0.07373 7 6 6V50 °C SFRS9 30.495 0.069 T287D / V433T50°C HPRT 29’810°96

[0853] 50 °C SFRS9 29.368 0.038 T287D / T467N50°C HPRT 30’562°77

[0854] 50 °C SFRS9 29.978 0.035 T T22S877DD / / AA550022SSTTOOPP50°CsHpPRRST92299'486g560Q'0Q748g

[0855]

[0856] T287D / L528Y50°C HPRT 30’519 70

[0857] 50 °C SFRS9 30.038 0.053 L333E / V433T50°C HPRT 30’394°’105

[0858] 50 °C SFRS9 29.813 0.03250°C HPRT 31’009°-070333 7 67N50 °C SFRS9 30.306 0.054 L L333333EE / / AA550U22SMTOUPP5500° oCC SHFPRRST93300’.42700000’.017043 L333E / L528Y 50 °C HPRT 30.474 0.07150 °C SFRS9 29.733 0.060 V433T / K435H V433T / K435H5

[0859] 500° oCC SHFPRRST

[0860] 9320’ 9.790928°’ 0.110101 V433T / T467N 50 °C HPRT 31.233 0.172

[0861] 50 °C SFRS9 30.507 0.046 V433T / G490A 50 °C HPRT 31.121 0.205

[0862] 50 °C SFRS9 30.475 0.089 V433T / A502STOP 50 °C HPRT 29.896 0.076

[0863] 50 °C SFRS9 29.219 0.070 V433T / L528Y 50 °C HPRT 31.168 0.123

[0864] 50 °C SFRS9 30.449 0.021 V433T / R629M 50 °C HPRT 31.449 0.072

[0865] 50 °C SFRS9 30.742 0.081 V433T / A646V 50 °C HPRT 30.895 0.136

[0866] 50 °C SFRS9 30.371 0.053 K K443355HH / / TT446677NN5500° oCC SHFPRRST933013.73321° 0.17358

[0867] K435H / A502STOP 50 °C HPRT 30.225 0.119 K435H / T 528Y50°C HPRT 31307°’042

[0868] 50 °C SFRS9 30.374 0.032 T467N / G490A 50 °C HPRT 31.588 0.082

[0869] 50 °C SFRS9 30.796 0.073 T TOT467N / AW502KSTTTOOP550O°.CC sHpPRRST930'358°Q-071

[0870] T467N / L528Y50°C HPRT 30’837°3867N / 5 850 °C SFRS9 30.106 0.082

[0871] T467N / R629M 50 °C HPRT 31.630 0.10950 °C SFRS9 31.006 0.101

[0872] T467N / A646V 50 °C HPRT 31.742 0.08050 °C SFRS9 31.002 0.028 G G449900AA / / AA550022SST 1OUPP5500°oCc SHFPRRST93300’.627681 °0 W362G490A / L528Y 50 °C HPRT 31.364 0.095

[0873] 50 °C SFRS9 30.679 0.089 1 L552288YY / / RR662299MM5500° oCC SHFPRRST93300’.934546 O0.088°2

[0874] L528Y / A646V 50 °C HPRT 31.682 0.13250 °C SFRS9 30.988 0.117DRO9AA / fTH2fi877nD / / TLW333EE5500° oCC SHFPRRST932093_78108°0-.004743

[0875] C157A / T287D / L333E 50 °C HPRT 29.961 0.03650 °C SFRS9 29.652 0.039R173E / T287D / L333E 50 °C HPRT 30.330 0.05350 °C SFRS9 29.999 0.075

[0876]

[0877] T287D / L333E / V433T 50 °C HPRT 30.063 0.06450 °C SFRS9 29.589 0.068 T92 TV / T T7 / T / I M50°C HPRT 29’9700.044 T287D / L333E / T467N50 oc SFRS9 29 529 0 014T T228877DD / / 1L 333333FE / / AA550022SSTTOOPP5500°oCc SHFPRRST92299355036°Q 06238T T2>s8,7Dr.. / , L333E / L528Y50°C HPRT 29-972°Q-093Mutant from App 50 °C HPRT 31.326 0.015 #20230031558 50 °C SFRS9 30.818 0.150

[0878] 50 °C HPRT 35.881 0.338

[0879]

[0880] 50 °C SFRS9 36.255 0.082

[0881] _ TABLE 18 _

[0882] Two-Step cDNA synthesis by single, double, and triple mutants of modified MMLV RTase. Data was generated via qPCR assay for HPRT OR SFRS9, as indicated by the “Target Name” column. Data is _ reported as Ct values. _

[0883] ,TZ Z * + cDNA Target Ct Ct MMLV RTase Variant „..

[0884] Temp Name Mean SD 55 °C HPRT 32.914 0.222

[0885]

[0886] A500Q 55 °C SFRS9 32.836 0.052

[0887] 55 °C HPRT 32.915 0.044 A619R

[0888] 55 °C SFRS9 32.602 0.075 55 °C HPRT 32.725 0.1966 955 °C HPRT 32.976 0.049 R9A / C157A 55 °C SFRS9 32.369 0.040

[0889] 55 °C SFRS9 32.399 0.178 C157A / R159P 55 °C HPRT 33.366 0.066

[0890] 55 °C SFRS9 32.572 0.087 C157A / R159S55°C HPRT 34’530°-232

[0891] 55 °C SFRS9 33.903 0.094 55 °C HPRT 31.814 0.179 55 °C SFRS9 31.341 0.084 C157A / T7R7D55°C HPRT 30’791°’083

[0892] 55 °C SFRS9 30.258 0.088 C157A / L333E 55 °C HPRT 31.249 0.080

[0893] 55 °C SFRS9 30.942 0.033 C157A / V433T 55 °C HPRT 31.344 0.060

[0894] 55 °C SFRS9 30.726 0.026 C C1I5577AA / / KK443355HH5„5°CSHFPRRST933212M551 00019041C C115477AA / / TT444677MN5„5”.Cc SHFPRRST93321 816549000Q9821

[0895]

[0896] C157A / G490A 55 °C HPRT 31.540 0.14055 °C SFRS9 31.058 0.072 C C157 / AA / / AA550U22STOUPP5555°OCC SHFPRRST93311 J74577004()7145E157A / T 528Y55°C HPRT 3 042°-°"

[0897] 55 °CSFRS9 31.701 0.042 55 °C HPRT 31.641 0.050 C157A / R629M55oC SFRS9 31 190 0.099

[0898] 55 °C HPRT 31.747 0.164C 57 7 6 6V55 °C SFRS9 31.084 0.083 R9A / R159P55°C HPRT 31684 0499

[0899] 55 °C SFRS9 31.025 0.029 R9A / R173F55°C HPRT 31 54°’2469A / 7355 °C SFRS9 31.042 0.031 R9A / T287D55°C HPRT 30’783°82

[0900] 55 °C SFRS9 30.181 0.091 55 °C HPRT 31.161 0.021

[0901]

[0902] 55 °CSFRS9 30.674 0.065 R9A / V433T55°C HPRT 30’871 60

[0903] 55 °C SFRS9 30.193 0.034 R R09AA / / KK443355HH5„5“ „Cc SHFPRRST93301'260909°0'014748

[0904] R9A / T467N55“C HPRT 32 134 0 229

[0905] 55 °C SFRS9 31.731 0.027 R R99AA / / GG449900AA 5555° oCC SHFPRRST931735 0Q409

[0906] 55 °C HPRT 33524 0032 R9A / A502STOP „SFRS9 31459R9A / 1 528Y55°C HPRT 31918°53

[0907] 55 °CSFRS9 31.608 0.125 55 °C HPRT 31.722 0.013 R9A / R629M55oC SFRS9 31 160 0.201

[0908] 55 °C HPRT 32.096 0.246

[0909]

[0910] 55 °C SFRS9 31.705 0.176 R R115599PP / / RR117733FE5555° oCC SHFPRRST9334138231130.515374 R159P / V433T55°C HPRT 30’944°81

[0911] 55 °C SFRS9 30.426 0.127 R159P / T467N55“C HPRT 31 967 0 049

[0912] 55 °C SFRS9 31.457 0.100 R R115599PP / / AA550022SSTTOOPP5555°oCc SHFPRRST93301882098 0040594i R159P / 1 528Y55°C HPRT 31784°85

[0913] 55 °C SFRS9 31.513 0.084 R159S / R173F

[0914] R159S / R173E5

[0915] 55

[0916] 5°

[0917] oC

[0918] C SHFPRRST

[0919] 93

[0920] 31

[0921] 2.7

[0922] 20

[0923] 22° 3 0.24920R159S / V433T55°C HPRT 31365 033855 °C SFRS9 31.063 0.092 R159S / T467N55°CHPRT 32.831 0.061

[0924] 55 °C SFRS9 32.791 0.092 55 °C HPRT 33 145 0 146 R159S / A502STOP „.c SPRS9 3 p523„ R R115599SS / / TL52288YY5555° oCC SHFPRRST93321’76016600306869R173F / T287D55°C HPRT 31304°37

[0925] 55 °C SFRS9 30.783 0.088 55 °C HPRT 31.158 0.038 R173E / L333EM„c spRS9 30 714 Q 033R173E / V433T55°C HPRT 31372°28

[0926] 55 °C SFRS9 31.120 0.013 R R117733FE / / KK443355HH5555° oCC SHFPRRST93311309021 003.00535 P 17 P / T / I 7M55°

[0927] R173E / T467N55oC

[0928] C SHFPRRST

[0929] 93

[0930] 31

[0931] 1-58

[0932] 514

[0933] 2003.14462 R173E / G490A55°C HPRT 32349 0328

[0934] 55 °C SFRS9 32.027 0.089 R R117733FE / / AA550022SSTTOOPP5555°oCc SHFPRRST93362320826003.04363 R173F / T 528Y55°C HPRT 32’000°’018

[0935] 55 °C SFRS9 31.729 0.069 R R117733FE / / RR662299MM5555° oCC SHFPRRST9332230999600.016655

[0936] 55 °C HPRT 31.983 0.165 R173E / A646V55oC SFRS9 31 535 0.044 T287D / V433T55°C HPRT 30’888°54T287D / V433T55 °C SFRS9 30.277 0.027 T T228877DD / / TT446677NN5„5°CSHFPRRST93311-5Q9o46°0-006862T T22S877DD / / AA550022SSTTOOPP55”CsHpPRRST

[0937]

[0938] 9330142952800009840T287D / 1 528Y55°C HPRT 31316 7°

[0939] 55 °C SFRS9 30.984 0.069 L333E / V433T55°C HPRT 31332°90

[0940] 55 °C SFRS9 30.609 0.046 T T7 / T / I '7'M55°C HPRT 31300 93333 7 67N55 °C SFRS9 31.195 0.085 L L333333EE / / AA550U22SMTOUPP5555° <>Cc SHFPRRST93321356561 00309100

[0941] 55 °C HPRT 31.505 0.082

[0942]

[0943] 55 °C SFRS9 31.003 0.055 V433T / K435H 55 °C HPRT 31.596 0.05655 °C SFRS9 30.923 0.177 V433T / T467N55“C HPRT 31'932 0J42

[0944] 55 °C SFRS9 31.390 0.042 55 °C HPRT 31 963 0 139 V433T / G490A

[0945] 55 °C SFRS9 31.401 0.111 55 °C HPRT 30554 0093 V433T / A502STOP

[0946] 55 °CSFRS9 30.180 0.026 V433T / L528Y55°C HPRT 31’824°22

[0947] 55 °C SFRS9 31.342 0.061 V433T / R629M55°C HPRT 31’982°’152

[0948] 55 °C SFRS9 31.530 0.067 V433T / A646V55°CHPRT 31.683 0.159

[0949] 55 °C SFRS9 31.087 0.089 K K443355HH / / TT446677NN5555° oCC SHFPRRST93321’064871°0’.019373

[0950] 55 °C HPRT 31 067 0 119 K435H / A502STOP „n

[0951] 55 °C SFRS9 30.603 0.109 K435H / T 528Y55°C HPRT 31’786°’109

[0952] 55 °C SFRS9 31.303 0.080 T467N / G490A55°C HPRT 32 83 0 50

[0953] 55 °C SFRS9 31.646 0.075 T Td4CT67NN / A5W0W2CTSTnOPP5„5°0C(,SHPPRRST93331 61fi85300009733T467N / 1 528Y55°C HPRT 31’710°’105

[0954] 55 °C SFRS9 31.073 0.042 55 °C HPRT 32.429 0.026 T467N / R629M55oC SFRS9 32 1160.164

[0955] 55 °C HPRT 32.280 0.187 T467N / A646V __

[0956] 55 °C SFRS9 31.915 0.062 G G449900AA / / AA550022SMTOUPP5555°oCc SHFPRRST93331’766171° Q17199G490A / L528Y55°C HPRT 32’042°’158

[0957] 55 °C SFRS9 31.573 0.086 L552288YY / / RR662299MM5555° oCC SHFPRRST93311’729074 ° 0.03528

[0958] 55 °C HPRT 32.406 0.163 L528Y / A646V55oC SFRS9 32 0100.042DOAfHfi7n / TWI355 °C HPRT 31.091 0.022 R9A / T287D / L333E55oC SFRS9 30_377 0 06i55 °C HPRT 30916 0019 C157A / T287D / L333E..0,

[0959] 55UC SFRS9 30.381 0.085 55 °C HPRT 31.156 0.074 R173E / T287D / L333E55oC SFRS9 30 661 0 063T T2x8n7Dw / Li u33w3EWm4t3T3Tr5M5°CsHpPRRST93300'J96905°-079

[0960]

[0961] T027D / T QQ- / T / I ™55°C HPRT 31’0380.099 T287D / L333E / T467N55 oc SFRS9 30 467 0 098

[0962] 55 °C HPRT 30653 0 119 T287D / L333E / A502STOP55,c SFRS9 M

[0963]

[0964] T^n / r nn / T ^v 55 °C HPRT 30.950 0.041 T287D / L333E / L528Y55 oc SFRS9 30 217 0 060Mutant from App 55 °C HPRT 32.370 0.060 #20230031558 55 °C SFRS9 32.141 0.057

[0965] 55 °C HPRT 38.902 1.147

[0966]

[0967] 55 °C SFRS9 38.411 1.168

[0968] _ TABLE 19 _

[0969] Two-Step cDNA synthesis by single, double, and triple mutants of modified MMLV RTase. Data was generated via qPCR assay for HPRT OR SFRS9, as indicated by the “Target Name” column. Data is reported as Ct values. Reverse Transcriptase Concentration = 5.2 nM final

[0970] • cDNA Target Ct Ct MMLV RTase Variant „ „ c r>

[0971] Temp Name Mean SD 60 °C HPRT 34.143 0.391 A500Q 60 °C HPRT 33.469 0.189

[0972] 60 °C HPRT 33.142 0.090 A619R

[0973] 60 °C HPRT 33.664 0.267 60 °C HPRT 33.406 0.441

[0974]

[0975] 6 960 °C HPRT 34.918 0.404 R9A / C157A60°C HPRT 32’331°-°74

[0976] 60 °C HPRT 31.068 0.229 C157A / R159P60°C HPRT 32’079°’186

[0977] 60 °C HPRT 31.838 0.124r

[0978] Cl,

[0979] 157

[0980] 7AA

[0981] / / R

[0982] RI,„

[0983] 159Ss6

[0984] 60

[0985] ()°

[0986] (>C

[0987] cH

[0988] HPPRRT

[0989] T3

[0990] 32.7

[0991] 6435°' 8 0.118127 C157A / R I73F

[0992] C157A / R173E6

[0993] 60

[0994] 0“

[0995] oC

[0996] CHHPPRRT

[0997] T3

[0998] 351'

[0999] .8

[1000] 15

[1001] 078°' 0.310529 C157A / T9R7D60°C HPRT 32’606°’151C157A / T287D60oCHPRT 32.208 0.075 C1 7A / T 333F60°C HPRT 34’780 3’694

[1002] 60 °C HPRT 31.496 0.087 C157A / V433T60 CHPRT 31.917 0.148

[1003] 60 °C HPRT 31.215 0.161 C157A / K435H60°

[1004] C157A / K435H60oC

[1005] CHHPPRRT

[1006] T3

[1007] 31’

[1008] 1.7

[1009] 13

[1010] 84°’ 3 0.117195ri 7A / T4 7NT60°C HPRT 31’8930.144C 57 7 67N60 °C HPRT 33.146 0.157 C157A / G490A60°C HPRT 32’005°’131L 37 / U 9U60 °C HPRT 36.234 0.728C C1n57 / AA / / AA550U22SMTOUPP660()°oCcHHPpRRTT332234720300’0149(8)CI S7A / I S9RY60°C HPRT 32’898°’166

[1011] 60 °C HPRT 32.160 0.372 60 °C HPRT 31.042 0.098 C157A / R629M60oCHPRT 32.221 0.045

[1012] 60 °C HPRT 33.969 0.452 C157A / A646VHPRT 32.417 0.119 R9A / R159P60°C HPRT 33364 0330

[1013] 60 °C HPRT 32.062 0.056 R9A / R173F60°C HPRT 33’849 0324

[1014] 60 °C HPRT 35.072 0.628 R9A / T287D60°C HPRT 32374 0’0689 7 8760 °C HPRT 31.919 0.105

[1015] 60 °C HPRT 32.046 0.157 60 °C HPRT 32.054 0.142 R9A / V433T60°C HPRT 32351 0386

[1016] 60 °C HPRT 33.152 0.190 R9A / K435H60” R9A / K435HC HHPPRRT

[1017] T3338.052i260i.403550 R9A / T467N60“C HPRT 33 051 0'216

[1018] 60 °C HPRT 33.482 0.199 R9A / G490A60°C HPRT 32572°’074

[1019] 60 °C HPRT 30.855 0.159 R9A / A502STOP60° R9A / A502STOP60oCCHHPPRRT

[1020] T33415.47538 005.42520 R9A / T 528Y60°C HPRT 32505°’062

[1021] 60 °C HPRT 31.506 0.062 60 °C HPRT 32.415 0.092 R9A / R629M60oCHPRT 38.455 0.656

[1022] 60 °C HPRT 32.149 0.171 R9A / A646VHPRT 31.617 0.246 R159P / R173F60°C HPRT 32323 0579

[1023] 60 °C HPRT 32.404 0.012 R159P / V433T60°C HPRT 34 646 0 412

[1024] 60 °C HPRT 32.453 0.255 R159P / T467N60°C HPRT 32'522°'268

[1025] 60 °C HPRT 32.356 0.094 R R115599PP / / AA550022SSTTOOPP6600°oCCHHPPRRTT33333.4316300’.058159 R159P / L528Y60°C HPRT 32558 0356

[1026] 60 °C HPRT 32.796 0.100 R159S / R173F60°C HPRT 36598 130159S / 7360 °C HPRT 32.210 0.177

[1027]

[1028] R159S / V433T 60 °C HPRT 33.492 0.28660 °C HPRT 33.197 0.082 R159S / T467N60°CHPRT 36.627 0.594

[1029] 60 °C HPRT 32.682 0.419 R1 599SS / / A 55002 SSTO 0P660()°oCcHHPpRRTT332333435600532278R159S / L528Y60°C HPRT 31543 0353

[1030] 60 °C HPRT 31.512 0.300 R173F / T287D60°C HPRT 31 81°’07873 7 8760 °C HPRT 30.760 0.082

[1031] 60 °C HPRT 31.088 0.102 R173E / L333E60oCHPRT 34.111 0.257 R173E / V433T60°C HPRT 3F008 0521

[1032] 60 °C HPRT 33.005 0.188 R R117733FE / / KK443355HH6600° oCC SHFPRRST9339319511005.39262 PIT

[1033] 73 7 / T / I

[1034] 6™

[1035] 7N 6

[1036] 60°

[1037] 0 °C

[1038] CSSFFRRS

[1039] S9

[1040] 93

[1041] 32

[1042] 25

[1043] .32

[1044] 678°- 0.226001 R173E / G490A60°C SFRS9 32’933°’047

[1045] 60 °C SFRS9 32.657 0.117 R R1I7733EE / / AA550022SSTTOOPP6600°„CcSSFFRRSS99334135943Q0Q o17161R R117733FE / / TL552288YY6600° oCCSSFFRRSS993301339599°0’006930R R.17733FE / / RR662299MM66°()”(>CcSSFFRRSS99331[397331°Q'°17Q21

[1046]

[1047] T. 60 °C SFRS9 32.125 0.20373 7 6 6V60 °C SFRS9 31.045 0.082 T287D / V433T60°C SFRS9 31596°’08787 7V 3360 °C SFRS9 31.838 0.117 T T228877DD / / TT446677NN6600° oCCSSFFRRSS993341323571 4 °’.009677 T T22S877DD / / AA550022SSTTOOPP6600“„CcSSFFRRSS993311'0M086°Q'015Q79T9R7D / T 59RY60°C SFRS9 30’439°’092

[1048] 60 °C SFRS9 31.257 0.189 60 °C SFRS9 30.383 0.108 60 °C SFRS9 31.058 0.098 T QQQT7 / ™™60°C SFRS9 32529 05 1 1333 7 67N60 °C SFRS9 31.279 0.086 L L333333EE / / AA550U22SMTOUPP6600°oCcSSFFRRSS9933213_87842 0Q3Q2546

[1049] 60 °C SFRS9 31.566 0.0546() oc SFRS9 32 ()19 0 154

[1050] V V443333TT / / KK443355HH66°()”(>CcSsFpRRSS993M1446136°'°53

[1051]

[1052] V433T / T467N60°C SFRS9 31'699 047760 °C SFRS9 31.397 0.062 V433T / G490A60°C SFRS9 31'795°'079

[1053] 60 °C SFRS9 32.366 0.164 V433T / A502STOP60°C SFRS9 3F61° °’252

[1054] 60 °C SFRS9 33.089 0.132 60 °C SFRS9 32.062 0.189 V433T / L528Y60oC SFRS9 32_072 0 061V433T / R629M60°C SFRS9 3F212°’°15

[1055] 60 °C SFRS9 31.302 0.024 V433T / A646V60“C SFRS9 31'730°'014

[1056] 60 °C SFRS9 31.442 0.134 K435H / T467N

[1057] K435H / T467N6

[1058] 60

[1059] 0°

[1060] oC

[1061] CSSFFRRS

[1062] S9

[1063] 93

[1064] 32’

[1065] 2.2

[1066] 372

[1067] 79°0’.027836 K K443355HH / / AA550022SSTTGOPP6600°oCcSSFFRRSS993322’916871°0’.303881 K K443355HH / / TL552288YY6600° oCCSSFFRRSS993321’599576°0’.007876

[1068]

[1069] T467N / G490A60°C SFRS9 30'214 0401

[1070] 60 °C SFRS9 31.379 0.121 T4A7NT / A sn9<? TnP

[1071] T467N / A502STOP6

[1072] 60

[1073] 0°

[1074] oC

[1075] cS

[1076] SFFRRS

[1077] S9

[1078] 93

[1079] 31

[1080] 1’

[1081] .4

[1082] 3799

[1083] 3°0’.013802 T467N / L528Y60°C SFRS9 3F009°’14167N / 5 860 °C SFRS9 31.861 0.138

[1084] 60 °C SFRS9 33.243 0.138 T467N / R629M60oC SFRS9 31 446 0.066

[1085] 60 °C SFRS9 31.149 0.130

[1086]

[1087] 67N / 6 6V6() oc SFRS931.923 0.132 G490A / A502STOP60°C SFRS9 31’864 0’092G490A / A502S 1 UP60 oc SFRS9 31_585Q Q91G U449y0UAA / / L^52Z88YY660()°oCcSSFFRRSS993321’J94358°0’21052()L L5522S8YY / / RR662299MM6600”,,CcSSFFRRSS993321 97 0Q'0o6798

[1088] 60 °C SFRS9 31.769 0.223 L528Y / A646VSRRS9 31 o n(.

[1089] P R09AA / / TT722877nD / / LT Q3Q3Q3PE6600°oCcSSFFRRSS99332F_7779°5° Q’219951G C115577AA / / TT228877DD / / 1L 333333FE6600° <>C SFRS9 3F47°O’°17

[1090]

[1091] c SFRS9 32 916 0 173

[1092] 60 °C SFRS9 32.544 0.303 R173E / T287D / L333E60<>c SFRS9 33 007 0 263T7Q TA / T60°cSFRS9 32.071 0.192 T287D / L333E / V433T60<>c SFRS9 31 802 0 010T287D / L333E / T467N 60 °C SFRS9 32.804 0.10260 °C SFRS9 30.924 0.103 T T228877DD / / LE333333EE / / AA550022SSTTOOPP6600°oCcSSFFRRSS993311’ _220352°Q’603922

[1093] T287D / E333E / E528Y6600°oCcSSFFRRSS993300-465805°0-007731Mutant from App 60 °C SFRS9 31.461 0.150 #20230031558 60 °C SFRS9 30.458 0.072 MMTVV’TT6600 ° °CCSSFFRRSS993372.87543 ° 0’.714341

[1094] _ TABLE 20 _

[1095] Two-Step cDNA synthesis by single, double, and triple mutants of modified MMLV RTase. Data was generated via qPCR assay for HPRT OR SFRS9, as indicated by the “Target Name” column. Data is reported as Ct values. Reverse Transcriptase Concentration = 5.2 nM final

[1096] MMLV RTase Variant cDNA Temp Target Name Ct Mean Ct SD 65 °C HPRT 35.278 0.102 A500Q 65 °C HPRT 34.512 0.299

[1097] 65 °C HPRT 34.580 0.264 A619R 65 °C HPRT 34.732 0.301 65 °C HPRT 34.044 0.347 65 °C HPRT 35.241 0.571 R9A / C157A65°C HPRT 33367 0329

[1098] 65 °C HPRT 31.749 0.093 C157A / R159P65°C HPRT 31819°’263

[1099] 65 °C HPRT 32.835 0.160 C157A / R159S65°C HPRT 33’787 0’404

[1100] 65 °C HPRT 33.794 0.268 65 °C HPRT 32.571 0.061 65 °C HPRT 36.043 0.609 C157A / T287D65°C HPRT 33’229°’251

[1101] 65 °C HPRT 32.799 0.381 C157A / T 777F65°C HPRT 32-4680.143

[1102] 65 °C HPRT 32.248 0.192 C157A / V433T65°C HPRT 33’168°’432

[1103] 65 °C HPRT 31.735 0.119 C157A / K435H C157A / K435H6

[1104] 655° oCCHHPPRRT

[1105] T3312.28221°’ 1 0.211170

[1106] C157A / T467N65"C HHPPRRT

[1107] T3343. O18I56003.34123

[1108] C157A / G490A65°C HPRT 32’846°’307

[1109] 65 °C HPRT 38.052 0.763

[1110]

[1111] C157A / A502STOP 65 °C HPRT 33.440 0.25665 °C HPRT 33.422 0.134 C157A / L528Y65°C HPRT 34’013 0365

[1112] 65 °C HPRT 33.366 0.360 65 °C HPRT 31.866 0.082C 57 7 6 965 °C HPRT 32.889 0.299

[1113] 65 °C HPRT 34.979 0.686C 57 7 6 6V65 °C HPRT 32.894 0.323 R9A / R159P65°C HPRT 33364°-3°9

[1114] 65 °C HPRT 33.127 0.410 R9A / R173F65°C HPRT 34’704°’563R9A / R173E65oCHPRT 35.448 0.446 R9A / T287D65°C HPRT 33’694 03009 7 8765 °C HPRT 32.840 0.068

[1115] 65"C HPRT 33-318 0-020R9A4.333EHPRT 34.142 0.481 R9A / V433T65°C HPRT 33’523 0369

[1116] 65 °C HPRT 34.931 0.513 R9A / K435H65°C HPRT 33 997 0 2569 7 3565 °C HPRT 38.781 0.863 R9A / T467N65“C HPRT 33 934°-290

[1117] 65 »C HPRT 34.447 0.309 R9A / G490A65°C HPRT 33337 0334

[1118] 65 °C HPRT 31.924 0.248 R9A / A502STOP R9A / A502STOP65°C HHPPRRT

[1119] T3335.094970 00.436968

[1120] R9A / 1 528Y65°C HPRT 32 895 03529 7 5 865 °C HPRT 32.995 0.247 T^nA ^™, 65 °C HPRT 33.910 0.220 R9A / R629M65oCHPRT 39.282 1.015 T.^ A, A <4< Ar65 °C HPRT 32.574 0.0449 7 6 6V65 °C HPRT 33.455 0.270 R159P / R173F65°C HPRT 34379 038459 7 7365 °C HPRT 33.328 0.352 R159P / V433T65°C HPRT 35388 0313

[1121] 65 °C HPRT 34.089 0.174 R159P / T467N65“C HPRT 34 212 0 269

[1122] 65 °C HPRT 33.629 0.500 65 °C HPRT 35 403 0 196 R159P / A502STOPHPRT^082 0^99 R159P / 1 528Y65° R159P / L528Y65oCCHHPPRRT

[1123] T33433.59226003.29865 R159S / R173F65°C HPRT 37316 032°59S / 7365 °C HPRT 33.492 0.110 R159S / V433T65” R159S / V433TC HHPPRRT

[1124] T3344.894178 00.454078

[1125]

[1126] R159S / T467N65°CHPRT 37.406 1.851 65 °C HPRT 33.635 0.452 R R 115599SS / / AA550022SSTTOOPP65” „CcHHPPRRTT3343.310148 00.614044 R159S / 1 528Y65°C HPRT 32434 0447

[1127] 65 °C HPRT 31.762 0.080 R R117733FB7TT22. R877DD65°C HHPPRRTT331L667780003.03374 n;,,,,,,, 65 »CHPRT 32-062°-100R173E / L333EHPRT 35.932 0.531 R173E / V433T65°C HPRT 31’804 0419

[1128] 65 °C HPRT 33.965 0.358 R R117733FE / / KK443355HH6655° oCC SHFPRRST93348 76423100.933636

[1129] 65 °C SFRS9 33.704 0.00873 7 67N65 °C SFRS9 33.249 0.190 R173E / G490A65°C SFRS9 33427 0411

[1130] 65 °C SFRS9 33.270 0.254 R173E / A502STOP65°C SFRS9 34418 0427R173E / A502STOP65 oc SFRS9 32 399Q069R173F / T 528Y65°C SFRS9 30’936 0415

[1131] 65 °C SFRS9 31.931 0.086 R R117733FE / / RR662299MM6655° oCCSSFFRRSS993321’.780379004.07368

[1132] 65 °C SFRS9 32.652 0.261 R173E / A646V65oC SFRS9 31 715 0.099 T287D / V433T65°C SFRS9 32496 0432

[1133] 65 °C SFRS9 32.383 0.109 T287D / T467N65°C SFRS9 3F70° °’017T287D / T467N65oC SFRS9 3F757 0.124 T T22S877DD / / AA550022SSTTOOPP6g55°C SSFFRRSS993311365721°Q'0193fi1

[1134] T T92R877DD / / TL5592R8YY6655° oCCSSFFRRSS993301’982303°0’013050

[1135] 65”CSFRS9 L333E / V433T3

[1136] 311-?05141<Q>-314333T_ / T.A-M65 °C SFRS9 32.761 0.082 L333E / T467N65oC SFRS9 31 562 0.092 L333E / A502STOP65°C SFRS9 32403 0423E333E / A5U2MUP65 oc SFRS9 32 412Q009T 333F / T 528Y65°C SFRS9 32404 040665 OC SFRS9 32 806 0 129V433T / K435H65°C SFRS9 32462 0430

[1137] 65 °C SFRS9 30.988 0.090

[1138]

[1139] V433T / T467N 65 °C SFRS9 32.288 0.23365 °C SFRS9 31.552 0.213 V433T / G490A65“C SFRS9 31'812°-109

[1140] 65 °C SFRS9 32.592 0.076 V433T / A502STOP65°C SFRS9 31594 0428

[1141] 65 °C SFRS9 33.810 0.355 V433T / L528Y65°C SFRS9 32’099 0430

[1142] 65 °C SFRS9 32.736 0.222 V433T / R629M65°C SFRS9 31497 0’040

[1143] 65 °C SFRS9 31.947 0.063 V433T / A646V65°C SFRS9 32352 038°

[1144] 65 °C SFRS9 31.771 0.115 K K443355HH / / TT446677NN6655° oCCSSFFRRSS9933224.82181° 0’.215572

[1145] K K443355HH / / AA550022SSTTGOPP6655°oCcSSFFRRSS993332’585849°’338

[1146] K K443355HH / / TL552288YY6655° oCCSSFFRRSS993322’916869004.06144 T467N / G490A65°C SFRS9 30'805°-050

[1147] 65 °C SFRS9 31.732 0.077 T4A7N / ASn7CTnp65"CSFRS9 T467N / A502STOPfi5 0(,SFRS93

[1148] 322JQ3198W0 1j7677

[1149] T T446677NN / / LT.5522S8YY65”C SSFFRRSS993321'95299100312320

[1150] 65 °C SFRS9 33.980 0.437 T467N / R629MspRS9 Q22

[1151] 65 °C SFRS9 32.249 0.046 67N / 6 6V65 OC SFRS9 32 868 0 ()99

[1152] G G449900AA / / AA550022SST 1O UPP6655°oCcSSFFRRSS993322405321 0414

[1153] G490A / L528Y65°C SFRS9 32563°’084

[1154] 65 °C SFRS9 33.005 0.189 L L552288YY / / RR662299MM6655° oCCSSFFRRSS9933234.75170°0’.035161

[1155] ^ 65 °C SFRS9 32.849 0.1075 8 7 6 6V65 °C SFRS9 32.978 0.198 ™ ™ 65 °C SFRS9 32.639 0.150 R9A / T287D / L333E65oC SFRS9 33 911 0 314C C115577AA / / TT228877DD / / 1L 333333FE6655° oCCSSFFRRSS993323’35440600424345

[1156] 65 °C SFRS9 33.329 0.206 R173E / T287D / L333E65<>c SFRS9 33 667 0 068

[1157] 65 °C SFRS9 32.527 0.132 T287D / L333E / V433T65<>c SFRS9 32 268 0 088

[1158] 65°C SFRS9 33.295 0.096 T287D / L333E / T467Nfi5 Q 076

[1159]

[1160] T T228877DD / / TL 333333EE / / AA550022SSTTOOPP6655°oCcSSFFRRSS993311’057846 0Q’012868Twn / T ^n / r ^v 65 °C

[1161] T287D / L333E / L528Y65 ocS

[1162] SFFRRS

[1163] S9

[1164] 93

[1165] 31

[1166] 1-0

[1167] 09

[1168] 21°- 101

[1169] 16

[1170] 23

[1171] 5Mutant from App 65 °C SLRS9 32.321 0.111 #20230031558 65 °C SLRS9 31.027 0.141

[1172] 65 °C SLRS9 33.175 0.202

[1173]

[1174] V’ 65 °C SLRS9 37.701 0.725

[1175] While the invention has been described with reference to an exemplary embodiment(s), it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment(s) disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.

[1176] DISCUSSION OF POSSIBLE EMBODIMENTS

[1177] The following are non-exclusive descriptions of possible embodiments of the present invention.

[1178] An embodiment of an isolated Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutant includes an amino acid sequence as set forth in any of SEQ ID NOs: 278-432.

[1179] An embodiment of a nucleotide includes a nucleotide encoding a foregoing isolated MMLV RTase mutant.

[1180] An embodiment of a cDNA includes a cDNA encoding a foregoing isolated MMLV RTase mutant.

[1181] An embodiment of a kit includes a kit comprising a foregoing isolated MMLV RTase mutant.

[1182] An embodiment of a composition includes a composition comprising a foregoing isolated MMLV RTase mutant.

[1183] A further embodiment of an isolated MMLV RTase variant includes a substitution at a position corresponding to least one of position 9, position 157, position 159, position 173, position 287, position 333, position 433, position 435, position 467, position 490, position 502, position 629, or position 646 of any of SEQ ID NOs: 275-277.The isolated MMLV RTase variant of the preceding paragraph can optionally include, additionally and / or alternatively, any one or more of the following features, configurations and / or additional components:

[1184] A further embodiment of the forgoing isolated MMLV RTase variant, wherein the variant comprises one or more substitutions selected from a group consisting of R9A, R9L, C157A, R159P, R159S, R173E, T287D, L333E, L333S, V433T, K435H, T467N, G490A, G490N, A502STOP, R629M, A646V, T287D+L333E, C157A+T287D, R9A+T287D, R9A+V433T, T287D+V433T, T287D+A502STOP, V433T+A502STOP, T287D+L333E+V433T, T287D+L333E+A502STOP, and T287D+L333E+L528Y.

[1185] A further embodiment of the forgoing isolated MMLV RTase variant, wherein the variant comprises one or more substitutions selected from a group consisting of R9A, C157A, R173E, V433T, T467N, A502STOP, L528Y, T287D+L333E, C157A+T287D, R9A+T287D, R9A+V433T, T287D+V433T, T287D+A502STOP, V433T+A502STOP, T287D+L333E+V433T, T287D+L333E+A502STOP, and T287D+L333E+L528Y.

[1186] An embodiment of a nucleotide includes a nucleotide encoding a foregoing isolated MMLV RTase variant.

[1187] An embodiment of a cDNA includes a cDNA encoding a foregoing isolated MMLV RTase variant.

[1188] An embodiment of a kit includes a kit comprising a foregoing isolated MMLV RTase variant.

[1189] An embodiment of a composition includes a composition comprising a foregoing isolated MMLV RTase variant.

[1190] An embodiment of an isolated prime editor fusion protein includes a nuclease domain and a reverse transcriptase domain having a sequence of an MMLV RTase mutant of a foregoing embodiment.

[1191] A method of synthesizing copy deoxyribonucleic acid (cDNA) includes providing a ribonucleic acid template and contacting the ribonucleic acid template with an MMLV RTase mutant of a foregoing embodiment to synthesize cDNA.

[1192] A method of gene editing includes providing a sample including a doublestranded target DNA sequence; and contacting the double-stranded target DNA sequence with a prime editor, the prime editor comprising: first domain comprising a nucleic acid programmable DNA binding protein; a second domain comprising a MMLV RTase mutant of a foregoing embodiment; and a prime editing guide RNA.The method of gene editing of the preceding paragraph can optionally include, additionally and / or alternatively, any one or more of the following features, configurations and / or additional components:

[1193] A further embodiment of the foregoing method of gene editing, wherein the nucleic acid programmable DNA binding protein comprises a nickase domain.

[1194] A method of performing reverse transcriptase-polymerase chain reaction (RT-PCR) including providing a ribonucleic acid template; contacting the ribonucleic acid template with an MMLV RTase variant of a foregoing embodiment to synthesize cDNA; and contacting the cDNA with a DNA polymerase to amplify the cDNA.

Claims

CLAIMS:

1. An isolated Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutant comprising an amino acid sequence as set forth in any of SEQ ID NOs: 278-432.

2. A nucleotide encoding the MMLV RTase mutant of claim 1.

3. A cDNA encoding the MMLV RTase mutant of claim 1.

4. A kit comprising the MMLV RTase mutant of claim 1.

5. A composition comprising the MMLV RTase mutant of claim 1.

6. An MMLV RTase variant comprising a substitution at a position corresponding to least one of position 9, position 157, position 159, position 173, position 287, position 333, position 433, position 435, position 467, position 490, position 502, position 629, or position 646 of any of SEQ ID NOs: 275-277.

7. The MMLV RTase variant of claim 6, wherein the variant comprises one or more substitutions selected from a group consisting of R9A, R9L, C157A, R159P, R159S, R173E, T287D, L333E, L333S, V433T, K435H, T467N, G490A, G490N, A502STOP, R629M, A646V, T287D+L333E, C157A+T287D, R9A+T287D, R9A+V433T, T287D+V433T, T287D+A502STOP, V433T+A502STOP, T287D+L333E+V433T, T287D+L333E+A502STOP, and T287D+L333E+L528Y.

8. The MMLV RTase variant of claim 6, wherein the variant comprises one or more substitutions selected from a group consisting of R9A, C157A, R173E, V433T, T467N, A502STOP, L528Y, T287D+L333E, C157A+T287D, R9A+T287D, R9A+V433T, T287D+V433T, T287D+A502STOP, V433T+A502STOP, T287D+L333E+V433T, T287D+L333E+A502STOP, and T287D+L333E+L528Y.

9. A nucleotide encoding the MMLV RTase mutant of claim 6.

10. A cDNA encoding the MMLV RTase mutant of claim 6.

11. A kit comprising the MMLV RTase mutant of claim 6.

12. A composition comprising the MMLV RTase mutant of claim 6.

13. An isolated prime editor fusion protein comprising:a nuclease domain; anda reverse transcriptase domain having a sequence of the MMLV RTase mutant of claim 6.

14. A method of synthesizing copy deoxyribonucleic acid (cDNA), the method comprising:providing a ribonucleic acid template; andcontacting the ribonucleic acid template with an MMLV RTase variant of claim 6 to synthesize cDNA.

15. A method of gene editing, the method comprising:providing a sample including a double- stranded target DNA sequence; and contacting the double- stranded target DNA sequence with a prime editor, the prime editor comprising:first domain comprising a nucleic acid programmable DNA binding protein;a second domain comprising the MMLV RTase variant of claim 6;anda prime editing guide RNA.

16. The method of claim 15, wherein the nucleic acid programmable DNA binding protein comprises a nickase domain.

17. A method of performing reverse transcriptase-polymerase chain reaction (RT-PCR), the method comprising:providing a ribonucleic acid template;contacting the ribonucleic acid template with an MMLV RTase variant of claim 6 to synthesize cDNA; andcontacting the cDNA with a DNA polymerase to amplify the cDNA.

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