LOCUS Exported 6303 bp ds-DNA circular SYN 27-SEP-2021 DEFINITION Mammalian expression of human MLH1dn (codon optimized). ACCESSION . VERSION . KEYWORDS pEF1a-hMLH1dn SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6303) TITLE Enhanced Prime Editing Systems Through Identification and Manipulation of Cellular Determinants of Editing Outcomes JOURNAL Unpublished REFERENCE 2 (bases 1 to 6303) AUTHORS . TITLE Direct Submission JOURNAL Exported Sep 27, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6303 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 193..1371 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 424..1362 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1319..1339 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 3663..3680 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(3703..3720) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3709..3933 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(4004..4020) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4004..4020) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4017..4039) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4028..4044 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(4052..4082) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4097..4118 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4235..4252) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4406..4994) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4486..4505) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5165..6025) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 5788..5807 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6026..6130) /gene="bla" /label=AmpR promoter primer_bind complement(6209..6228) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg 61 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actgcaaaga 121 tggataaagt tttaaacaga gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa 181 ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 241 gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta 301 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg 361 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca 421 caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc 481 gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt 541 ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga 601 gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc 661 tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg 721 ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg 781 gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg 841 cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct 901 gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc 961 cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc 1021 tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa 1081 agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc 1141 aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg 1201 ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg 1261 cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc 1321 aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg acgtacggga 1381 tcctctagag ccaccatgag cttcgttgct ggagtcatcc ggagactgga cgagacagtg 1441 gtgaacagaa ttgccgccgg cgaggtgatc cagagacctg ccaatgcaat aaaggagatg 1501 atcgagaact gtctggacgc caagtccaca agcattcagg tgatcgtgaa ggagggcgga 1561 ctgaagctga tccagatcca agacaacggc acaggcatca gaaaggaaga tctggacatc 1621 gtgtgtgaac ggttcaccac atctaagctg cagtcttttg aggatctggc ctctatcagt 1681 acctacggct tcagaggcga ggccctggcc agcatcagcc acgtggccca tgtgaccatc 1741 accaccaaaa ccgccgacgg caaatgcgct tatcgcgcta gctacagcga cggcaagctg 1801 aaagccccgc caaagccttg cgccggcaac cagggtacac agataacagt ggaggatctg 1861 ttctacaaca tcgccacccg gagaaaggcc ctgaaaaatc ccagcgagga gtacggcaag 1921 atcctggaag tcgtcggcag atactccgtg cacaacgccg gaatcagctt tagcgtaaag 1981 aagcagggag aaaccgtggc cgatgtgcgc accctgccaa atgccagcac cgtggataac 2041 atcagaagca ttttcggaaa tgccgtgtcc agagaactga tcgagatcgg ctgcgaagat 2101 aagaccctgg cttttaagat gaacggctac atcagcaacg ccaattactc tgtgaagaag 2161 tgcatctttc ttctgttcat caaccacaga ctggtggaaa gcaccagcct gcggaaagcc 2221 atcgagacag tgtacgccgc ctacctgcct aagaacaccc accccttcct gtacctgagc 2281 ctcgagatca gccctcagaa cgtggacgtc aatgtgcatc ctacaaagca cgaggtgcac 2341 ttcctgcacg aggaaagcat cctggaaaga gtgcagcagc acattgagag caagctgctg 2401 ggctctaaca gcagcagaat gtacttcaca cagaccctgc tgcctggcct ggccggcccc 2461 tcaggcgaaa tggttaagtc cacaacctct ctgacctcat ccagcaccag cggttcttcc 2521 gataaggtgt acgcccacca gatggtgcgg accgactctc gggagcagaa gctggacgcc 2581 tttctgcaac ctctgagcaa acctctgagc tctcagcctc aggccatcgt gaccgaggac 2641 aagacagata tctcctccgg ccgtgccaga cagcaggacg aagaaatgct cgagctgcca 2701 gctcctgccg aggtggccgc caagaaccag agcctggagg gagataccac aaagggcacc 2761 agcgaaatga gcgagaagcg gggccctacc tccagcaacc ccagaaaacg gcaccgggag 2821 gacagcgacg tggaaatggt ggaggacgac agcagaaagg aaatgacagc cgcttgtacc 2881 cctagaagaa gaatcatcaa cctgacctcc gtgctgagcc tgcaggagga gatcaacgag 2941 cagggccacg aggtgctgag agagatgctg cacaatcaca gcttcgtggg ctgcgtgaac 3001 cctcaatggg ccctggctca gcatcaaaca aagctgtacc tgctgaacac caccaagctg 3061 agcgaagagc tgttctacca gatcctcatc tacgacttcg ccaacttcgg cgtgctacgc 3121 ctgagcgagc ccgcccctct gtttgacctg gccatgctgg ctctggatag cccagaaagc 3181 ggctggacag aagaggacgg acctaaagag gggctggctg aatacatcgt ggagttcctg 3241 aagaaaaagg ccgagatgct ggccgactac ttttctctgg aaatcgacga ggaaggcaac 3301 ctgatcggcc tgcctctgct gatcgataac tacgtgcctc ccctggaagg cctgcccatc 3361 ttcatcctga gactggctac agaggtgaac tgggacgagg aaaaggaatg cttcgagtct 3421 ctgagcaagg agtgcgccat gttctatagc atcagaaaac agtacatctc tgaagagagc 3481 actctgtctg gccagcagag tgaagtgccc ggaagcatcc ccaacagctg gaagtggacc 3541 gtggaacaca tcgtgtacaa ggccctgcgg agccacattc tccctcctaa gcacttcacc 3601 gaggacggca acatcctgca gctggccaac ctgcccgacc tttataaggt tttctaaccg 3661 gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact gtgccttcta 3721 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 3781 ctcccactgt cctttcctaa taaaatgaga aaattgcatc gcattgtctg agtaggtgtc 3841 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata 3901 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg 3961 gctcgatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg 4021 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 4081 aagcctaggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 4141 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 4201 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 4261 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 4321 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 4381 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 4441 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 4501 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 4561 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 4621 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 4681 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 4741 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 4801 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 4861 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 4921 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 4981 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacact cagtggaacg 5041 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 5101 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 5161 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 5221 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 5281 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 5341 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 5401 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 5461 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 5521 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 5581 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 5641 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 5701 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 5761 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 5821 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 5881 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 5941 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 6001 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 6061 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 6121 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatcgat 6181 ctcccgatcc cctagggtcg actctcagta caatctgctc tgatgccgca tagttaagcc 6241 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag 6301 cta //