LOCUS Exported 8811 bp ds-DNA circular SYN 27-SEP-2021 DEFINITION Mammalian expression of SaCas9 prime editor 2. ACCESSION . VERSION . KEYWORDS pCMV-SaPE2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8811) TITLE Enhanced Prime Editing Systems Through Identification and Manipulation of Cellular Determinants of Editing Outcomes JOURNAL Unpublished REFERENCE 2 (bases 1 to 8811) AUTHORS . TITLE Direct Submission JOURNAL Exported Sep 27, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8811 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 64..443 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 444..647 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 598..618 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 689..708 /label=T7 /note="T7 promoter, forward primer" promoter 689..707 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 757..777 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 6109..6129 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 6138..6155 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(6178..6195) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6184..6408 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(6479..6495) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6479..6495) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6492..6514) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6503..6519 /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(6527..6557) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6572..6593 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6710..6727) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6881..7469) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6961..6980) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7640..8500) /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 8263..8282 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8501..8605) /gene="bla" /label=AmpR promoter primer_bind complement(8684..8703) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc 61 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 121 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 181 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 241 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 301 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 361 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 421 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 481 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 541 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 601 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc 661 gtcagatccg ctagagatcc gcggccgcta atacgactca ctatagggag agccgccacc 721 atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtcggg 781 aagcgaaatt acattctggg gctggacatt ggcattacat cagtgggcta tggcatcatt 841 gactacgaga caagggacgt gatcgacgcc ggcgtgagac tgttcaagga ggccaacgtg 901 gagaacaatg agggccggag atccaagagg ggagcaaggc gcctgaagcg gagaaggcgc 961 cacagaatcc agagagtgaa gaagctgctg ttcgattaca acctgctgac cgaccactcc 1021 gagctgtctg gcatcaatcc ttatgaggcc agagtgaagg gcctgtccca gaagctgtct 1081 gaggaggagt ttagcgccgc cctgctgcac ctggcaaaga ggagaggcgt gcacaacgtg 1141 aatgaggtgg aggaggacac cggcaacgag ctgtccacaa aggagcagat cagccgcaat 1201 tccaaggccc tggaggagaa gtatgtggcc gagctgcagc tggagcggct gaagaaggat 1261 ggcgaggtga ggggctccat caatcgcttc aagacctctg actacgtgaa ggaggccaag 1321 cagctgctga aggtgcagaa ggcctaccac cagctggatc agtcctttat cgatacatat 1381 atcgacctgc tggagacaag gcgcacatac tatgagggac caggagaggg ctctcccttc 1441 ggctggaagg acatcaagga gtggtacgag atgctgatgg gccactgcac ctattttcca 1501 gaggagctga gaagcgtgaa gtacgcctat aacgccgatc tgtacaacgc cctgaatgac 1561 ctgaacaacc tggtcatcac cagggatgag aacgagaagc tggagtacta tgagaagttc 1621 cagatcatcg agaacgtgtt caagcagaag aagaagccta cactgaagca gatcgccaag 1681 gagatcctgg tgaacgagga ggacatcaag ggctaccgcg tgacctccac aggcaagcca 1741 gagttcacca atctgaaggt gtatcacgat atcaaggaca tcacagcccg gaaggagatc 1801 atcgagaacg ccgagctgct ggatcagatc gccaagatcc tgaccatcta tcagagctcc 1861 gaggacatcc aggaggagct gaccaacctg aatagcgagc tgacacagga ggagatcgag 1921 cagatcagca atctgaaggg ctacaccggc acacacaacc tgagcctgaa ggccatcaat 1981 ctgatcctgg atgagctgtg gcacacaaac gacaatcaga tcgccatctt taaccggctg 2041 aagctggtgc caaagaaggt ggacctgtcc cagcagaagg agatcccaac cacactggtg 2101 gacgatttca tcctgtctcc cgtggtgaag cggagcttca tccagagcat caaagtgatc 2161 aacgccatca tcaagaagta cggcctgccc aatgatatca tcatcgagct ggccagggag 2221 aagaactcca aggacgccca gaagatgatc aatgagatgc agaagaggaa ccgccagacc 2281 aatgagcgga tcgaggagat catcagaacc acaggcaagg agaacgccaa gtacctgatc 2341 gagaagatca agctgcacga tatgcaggag ggcaagtgtc tgtattctct ggaggccatc 2401 cctctggagg acctgctgaa caatccattc aactacgagg tggatcacat catcccccgg 2461 agcgtgagct tcgacaattc ttttaacaat aaggtgctgg tgaagcagga ggaggccagc 2521 aagaagggca ataggacccc tttccagtac ctgtctagct ccgattctaa gatcagctac 2581 gagacattca agaagcacat cctgaatctg gccaagggca agggccgcat cagcaagacc 2641 aagaaggagt acctgctgga ggagcgggac atcaacagat tctccgtgca gaaggacttc 2701 atcaaccgga atctggtgga caccagatac gccacacgcg gcctgatgaa tctgctgcgg 2761 tcttatttca gagtgaacaa tctggatgtg aaggtgaaga gcatcaacgg cggcttcacc 2821 tcctttctgc ggagaaagtg gaagtttaag aaggagcgca acaagggcta taagcaccac 2881 gccgaggatg ccctgatcat cgccaatgcc gacttcatct ttaaggagtg gaagaagctg 2941 gacaaggcca agaaagtgat ggagaaccag atgttcgagg agaagcaggc cgagagcatg 3001 cccgagatcg agacagagca ggagtacaag gagattttca tcacacctca ccagatcaag 3061 cacatcaagg acttcaagga ctacaagtat tctcacaggg tggataagaa gcccaaccgc 3121 gagctgatca atgacaccct gtatagcaca cggaaggacg ataagggcaa taccctgatc 3181 gtgaacaatc tgaacggcct gtacgacaag gataatgaca agctgaagaa gctgatcaac 3241 aagtctcccg agaagctgct gatgtaccac cacgatcctc agacatatca gaagctgaag 3301 ctgatcatgg agcagtacgg cgacgagaag aacccactgt ataagtacta tgaggagaca 3361 ggcaactacc tgacaaagta tagcaagaag gataatggcc ccgtgatcaa gaagatcaag 3421 tactatggca acaagctgaa tgcccacctg gacatcaccg acgattaccc taactctcgc 3481 aataaggtgg tgaagctgag cctgaagcca taccggttcg acgtgtacct ggacaacggc 3541 gtgtataagt ttgtgacagt gaagaatctg gatgtgatca agaaggagaa ctactatgag 3601 gtgaacagca agtgctacga ggaggccaag aagctgaaga agatcagcaa ccaggccgag 3661 ttcatcgcct ctttttacaa caatgacctg atcaagatca atggcgagct gtatagagtg 3721 atcggcgtga acaatgatct gctgaacaga atcgaagtga atatgatcga catcacctac 3781 agggagtatc tggagaacat gaatgataag aggccccctc gcatcatcaa gaccatcgcc 3841 tctaagacac agagcatcaa gaagtacagc acagacatcc tggggaacct gtatgaagtc 3901 aagagcaaga aacatcctca gattatcaag aaaggctctg gaggatctag cggaggatcc 3961 tctggcagcg agacaccagg aacaagcgag tcagcaacac cagagagcag tggcggcagc 4021 agcggcggca gcagcaccct aaatatagaa gatgagtatc ggctacatga gacctcaaaa 4081 gagccagatg tttctctagg gtccacatgg ctgtctgatt ttcctcaggc ctgggcggaa 4141 accgggggca tgggactggc agttcgccaa gctcctctga tcatacctct gaaagcaacc 4201 tctacccccg tgtccataaa acaatacccc atgtcacaag aagccagact ggggatcaag 4261 ccccacatac agagactgtt ggaccaggga atactggtac cctgccagtc cccctggaac 4321 acgcccctgc tacccgttaa gaaaccaggg actaatgatt ataggcctgt ccaggatctg 4381 agagaagtca acaagcgggt ggaagacatc caccccaccg tgcccaaccc ttacaacctc 4441 ttgagcgggc tcccaccgtc ccaccagtgg tacactgtgc ttgatttaaa ggatgccttt 4501 ttctgcctga gactccaccc caccagtcag cctctcttcg cctttgagtg gagagatcca 4561 gagatgggaa tctcaggaca attgacctgg accagactcc cacagggttt caaaaacagt 4621 cccaccctgt ttaatgaggc actgcacaga gacctagcag acttccggat ccagcaccca 4681 gacttgatcc tgctacagta cgtggatgac ttactgctgg ccgccacttc tgagctagac 4741 tgccaacaag gtactcgggc cctgttacaa accctaggga acctcgggta tcgggcctcg 4801 gccaagaaag cccaaatttg ccagaaacag gtcaagtatc tggggtatct tctaaaagag 4861 ggtcagagat ggctgactga ggccagaaaa gagactgtga tggggcagcc tactccgaag 4921 acccctcgac aactaaggga gttcctaggg aaggcaggct tctgtcgcct cttcatccct 4981 gggtttgcag aaatggcagc ccccctgtac cctctcacca aaccggggac tctgtttaat 5041 tggggcccag accaacaaaa ggcctatcaa gaaatcaagc aagctcttct aactgcccca 5101 gccctggggt tgccagattt gactaagccc tttgaactct ttgtcgacga gaagcagggc 5161 tacgccaaag gtgtcctaac gcaaaaactg ggaccttggc gtcggccggt ggcctacctg 5221 tccaaaaagc tagacccagt agcagctggg tggccccctt gcctacggat ggtagcagcc 5281 attgccgtac tgacaaagga tgcaggcaag ctaaccatgg gacagccact agtcattctg 5341 gccccccatg cagtagaggc actagtcaaa caaccccccg accgctggct ttccaacgcc 5401 cggatgactc actatcaggc cttgcttttg gacacggacc gggtccagtt cggaccggtg 5461 gtagccctga acccggctac gctgctccca ctgcctgagg aagggctgca acacaactgc 5521 cttgatatcc tggccgaagc ccacggaacc cgacccgacc taacggacca gccgctccca 5581 gacgccgacc acacctggta cacggatgga agcagtctct tacaagaggg acagcgtaag 5641 gcgggagctg cggtgaccac cgagaccgag gtaatctggg ctaaagccct gccagccggg 5701 acatccgctc agcgggctga actgatagca ctcacccagg ccctaaagat ggcagaaggt 5761 aagaagctaa atgtttatac tgatagccgt tatgcttttg ctactgccca tatccatgga 5821 gaaatataca gaaggcgtgg gtggctcaca tcagaaggca aagagatcaa aaataaagac 5881 gagatcttgg ccctactaaa agccctcttt ctgcccaaaa gacttagcat aatccattgt 5941 ccaggacatc aaaagggaca cagcgccgag gctagaggca accggatggc tgaccaagcg 6001 gcccgaaagg cagccatcac agagactcca gacacctcta ccctcctcat agaaaattca 6061 tcaccctctg gcggctcaaa aagaaccgcc gacggcagcg aattcgagcc caagaagaag 6121 aggaaagtct aaccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct 6181 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 6241 ccctggaagg tgccactccc actgtccttt cctaataaaa tgagaaaatt gcatcgcatt 6301 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg 6361 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg 6421 aaagaaccag ctggggctcg ataccgtcga cctctagcta gagcttggcg taatcatggt 6481 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg 6541 gaagcataaa gtgtaaagcc tagggtgcct aatgagtgag ctaactcaca ttaattgcgt 6601 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 6661 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 6721 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 6781 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 6841 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 6901 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 6961 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 7021 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 7081 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 7141 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 7201 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 7261 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 7321 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 7381 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 7441 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 7501 acactcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 7561 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 7621 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 7681 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 7741 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 7801 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 7861 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 7921 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 7981 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 8041 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 8101 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 8161 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 8221 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 8281 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 8341 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 8401 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 8461 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 8521 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 8581 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg 8641 gatcgggaga tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg 8701 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 8761 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg a //