LOCUS Exported 5693 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION donor vector for AAVS1 targeting (neomycin selection) with multiple cloning site. ACCESSION . VERSION . KEYWORDS pAAVS1-Nst-MCS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5693) AUTHORS Oceguera-Yanez F, Kim SI, Matsumoto T, Tan GW, Xiang L, Hatani T, Kondo T, Ikeya M, Yoshida Y, Inoue H, Woltjen K TITLE Engineering the AAVS1 locus for consistent and scalable transgene expression in human iPSCs and their differentiated derivatives. JOURNAL Methods. 2015 Dec 18. pii: S1046-2023(15)30181-X. doi: 10.1016/j.ymeth.2015.12.012. PUBMED 26707206 REFERENCE 2 (bases 1 to 5693) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5693 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 21..125 /gene="bla" /label=AmpR promoter CDS 126..986 /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 complement(344..363) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1157..1745 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1646..1665 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1899..1916 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2033..2054 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2069..2099 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2107..2123 /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)." primer_bind 2112..2134 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2131..2147 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2131..2147 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 2168..2186 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" misc_feature 2199..3002 /label=HA-L /note="left homology arm from the adeno-associated virus integration site (AAVS1) within intron 1 of the human PPP1R12C gene" misc_feature 3009..3034 /label=SA /note="splice acceptor site" CDS 3058..3111 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 3127..3921 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3181..3200) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3791..3810 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(3941..3958) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3947..4171 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" misc_feature 4374..4903 /label=HA-R /note="right homology arm from the adeno-associated virus integration site (AAVS1) within intron 1 of the human PPP1R12C gene" primer_bind complement(5049..5068) /label=T7 /note="T7 promoter, forward primer" promoter complement(5050..5068) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5075..5092) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5075..5091) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5084..5106) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 5233..5688 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5320..5339) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5530..5551 /label=F1ori-F /note="F1 origin, forward primer" ORIGIN 1 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 61 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 121 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 241 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 301 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 361 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 421 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 541 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 661 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 721 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 781 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 841 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 961 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 1021 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 1081 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 1141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 1201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 1261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 1321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 1381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 1441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 1501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 1561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 1621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 1681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 1741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 1801 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 1861 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 1921 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 1981 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga 2041 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt 2101 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca 2161 agctcgaaat taaccctcac taaagggaac aaaagctgtg ctttctctga ccagcattct 2221 ctcccctggg cctgtgccgc tttctgtctg cagcttgtgg cctgggtcac ctctacggct 2281 ggcccagatc cttccctgcc gcctccttca ggttccgtct tcctccactc cctcttcccc 2341 ttgctctctg ctgtgttgct gcccaaggat gctctttccg gagcacttcc ttctcggcgc 2401 tgcaccacgt gatgtcctct gagcggatcc tccccgtgtc tgggtcctct ccgggcatct 2461 ctcctccctc acccaacccc atgccgtctt cactcgctgg gttccctttt ccttctcctt 2521 ctggggcctg tgccatctct cgtttcttag gatggccttc tccgacggat gtctcccttg 2581 cgtcccgcct ccccttcttg taggcctgca tcatcaccgt ttttctggac aaccccaaag 2641 taccccgtct ccctggcttt agccacctct ccatcctctt gctttctttg cctggacacc 2701 ccgttctcct gtggattcgg gtcacctctc actcctttca tttgggcagc tcccctaccc 2761 cccttacctc tctagtctgt gctagctctt ccagccccct gtcatggcat cttccagggg 2821 tccgagagct cagctagtct tcttcctcca acccgggccc ctatgtccac ttcaggacag 2881 catgtttgct gcctccaggg atcctgtgtc cccgagctgg gaccacctta tattcccagg 2941 gccggttaat gtggctctgg ttctgggtac ttttatctgt cccctccacc ccacagtggg 3001 gcaagcttct gacctcttct cttcctccca cagggcctcg agagatctgg cagcggagag 3061 ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atcccggccc taggctcgag 3121 ggtaccatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg 3181 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg 3241 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat 3301 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca 3361 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg 3421 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat 3481 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa 3541 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg 3601 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg 3661 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg 3721 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat 3781 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac 3841 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc 3901 cttcttgacg agttcttctg agtttaaacc cgctgatcag cctcgactgt gccttctagt 3961 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 4021 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 4081 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc 4141 aggcatgctg gggatgcggt gggctctatg gactagtatt aattaaatct agaagtcgac 4201 agtactaagc tttgacagaa aagccccatc cttaggcctc ctccttccta gtctcctgat 4261 attgggtcta acccccacct cctgttaggc agattcctta tctggtgaca cacccccatt 4321 tcctggagcc atctctctcc ttgccagaac ctctaaggtt tgcttacgat ggagccagag 4381 aggatcctgg gagggagagc ttggcagggg gtgggaggga agggggggat gcgtgacctg 4441 cccggttctc agtggccacc ctgcgctacc ctctcccaga acctgagctg ctctgacgcg 4501 gctgtctggt gcgtttcact gatcctggtg ctgcagcttc cttacacttc ccaagaggag 4561 aagcagtttg gaaaaacaaa atcagaataa gttggtcctg agttctaact ttggctcttc 4621 acctttctag tccccaattt atattgttcc tccgtgcgtc agttttacct gtgagataag 4681 gccagtagcc agccccgtcc tggcagggct gtggtgagga ggggggtgtc cgtgtggaaa 4741 actccctttg tgagaatggt gcgtcctagg tgttcaccag gtcgtggccg cctctactcc 4801 ctttctcttt ctccatcctt ctttccttaa agagtcccca gtgctatctg ggacatattc 4861 ctccgcccag agcagggtcc cgcttcccta aggccctgct ctgggcttct gggtttgagt 4921 ccttggcaag cccaggagag gcgctcaggc ttccctgtcc cccttcctcg tccaccatct 4981 catgcccctg gctctcctgc cccttcccta caggggttcc tggctctgct ctagcgatcg 5041 ccaattcgcc ctatagtgag tcgtattaca attcactggc cgtcgtttta caacgtcgtg 5101 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 5161 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 5221 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 5281 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 5341 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 5401 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 5461 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 5521 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 5581 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 5641 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtg //