LOCUS Exported 8682 bp ds-DNA linear SYN 18-MAY-2021 DEFINITION Tetracycline inducible expression of guide RNA targeted to AAVS1 locus. ACCESSION . VERSION . KEYWORDS pAAV-Puro_siKO SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8682) AUTHORS Bertero A, Pawlowski M, Ortmann D, Snijders K, Yiangou L, Cardoso de Brito M, Brown S, Bernard WG, Cooper JD, Giacomelli E, Gambardella L, Hannan NR, Iyer D, Sampaziotis F, Serrano F, Zonneveld MC, Sinha S, Kotter M, Vallier L TITLE Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs. JOURNAL Development. 2016 Dec 1;143(23):4405-4418. PUBMED 27899508 REFERENCE 2 (bases 1 to 8682) AUTHORS . TITLE Direct Submission JOURNAL Exported May 18, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8682 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 225..247 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 309..328 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" CDS 372..392 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" polyA_signal 1046..1157 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" misc_feature 1287..2123 /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(2157..2176) /label=T7 /note="T7 promoter, forward primer" promoter complement(2158..2176) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2183..2200) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2183..2199) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2192..2214) /label=M13/pUC Forward /note="In lacZ gene" CDS 2337..2639 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 2518..2537 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 2988..3782 /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 GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3042..3061) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3652..3671 /label=Neo-F /note="Neomycin resistance gene, forward primer" CDS complement(4032..4892) /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 4655..4674 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5016..5604 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5505..5524 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5758..5775 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5892..5913 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5928..5958 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5966..5982 /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 5971..5993 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5990..6006 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5990..6006 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 6027..6045 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" misc_feature 6080..6883 /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 6890..6915 /label=SA /note="splice acceptor site" CDS 6939..6992 /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 7002..7601 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(7002..7021) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 7498..7518 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 7639..7863 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind 8007..8026 /label=H1 /note="Human H1 promoter, forward primer" protein_bind 8072..8090 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" misc_RNA 8125..8200 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 8279..8582 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg 61 caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg 121 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg 181 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg 241 tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca 301 gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattaattcg 361 gatccaccat gcccaagaaa aagcggaagg tgtcccggct ggacaagagc aaagtgatca 421 acagcgccct ggaactgctg aacgaagtgg gcatcgaggg cctgaccacc cggaagctgg 481 cccagaaact gggcgtggaa cagcccaccc tgtactggca cgtgaagaac aagcgggccc 541 tgctggacgc cctggccatc gagatgctgg accggcacca cacacacttt tgccccctgg 601 aaggcgaaag ctggcaggac ttcctgcgga acaacgccaa gagcttcaga tgcgccctgc 661 tgagccaccg ggacggcgcc aaagtgcacc tgggcaccag acccaccgag aagcagtacg 721 agacactgga aaaccagctg gccttcctgt gccagcaggg cttcagcctg gaaaacgccc 781 tgtacgccct gagcgccgtg ggccacttta ccctgggctg cgtgctggaa gatcaggaac 841 accaggtcgc caaagaggaa agagagacac ccaccaccga cagcatgccc cccctgctga 901 gacaggccat cgagctgttc gatcatcaag gcgccgagcc cgccttcctg ttcggcctgg 961 aactgatcat ctgcggcctc gagaagcagc tgaagtgcga gagcggctcc cccagatgaa 1021 cgcgtagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc 1081 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa 1141 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg 1201 gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg gggtctcgag 1261 ggggggcccg gacagtacta agctttacta gggacaggat tggtgacaga aaagccccat 1321 ccttaggcct cctccttcct agtctcctga tattgggtct aacccccacc tcctgttagg 1381 cagattcctt atctggtgac acacccccat ttcctggagc catctctctc cttgccagaa 1441 cctctaaggt ttgcttacga tggagccaga gaggatcctg ggagggagag cttggcaggg 1501 ggtgggaggg aaggggggga tgcgtgacct gcccggttct cagtggccac cctgcgctac 1561 cctctcccag aacctgagct gctctgacgc ggctgtctgg tgcgtttcac tgatcctggt 1621 gctgcagctt ccttacactt cccaagagga gaagcagttt ggaaaaacaa aatcagaata 1681 agttggtcct gagttctaac tttggctctt cacctttcta gtccccaatt tatattgttc 1741 ctccgtgcgt cagttttacc tgtgagataa ggccagtagc cagccccgtc ctggcagggc 1801 tgtggtgagg aggggggtgt ccgtgtggaa aactcccttt gtgagaatgg tgcgtcctag 1861 gtgttcacca ggtcgtggcc gcctctactc cctttctctt tctccatcct tctttcctta 1921 aagagtcccc agtgctatct gggacatatt cctccgccca gagcagggtc ccgcttccct 1981 aaggccctgc tctgggcttc tgggtttgag tccttggcaa gcccaggaga ggcgctcagg 2041 cttccctgtc ccccttcctc gtccaccatc tcatgcccct ggctctcctg ccccttccct 2101 acaggggttc ctggctctgc tctaagggcg aattcgcggc cgctaaattc aattcgccct 2161 atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 2221 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 2281 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctatac gtacggcagt 2341 ttaaggttta cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg 2401 atattattga cacgccgggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt 2461 cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca 2521 tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa gtggctgatc 2581 tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataaa 2641 tgtcaggcat gagattatca aaaaggatct tcacctagat ccttttcacg tagaaagcca 2701 gtccgcagaa acggtgctga ccccggatga atgtcagcta ctgggctatc tggacaaggg 2761 aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag 2821 actgggcggt tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta 2881 aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc 2941 gcaggggatc aagctctgat caagagacag gatgaggatc gtttcgcatg attgaacaag 3001 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg 3061 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc 3121 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag 3181 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca 3241 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat 3301 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata 3361 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac 3421 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc 3481 tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg 3541 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg 3601 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta 3661 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg 3721 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct 3781 gaattattaa cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta 3841 tttcacaccg catcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 3901 tttttctaaa tacattcaaa tatgtatccg ctcatgagat tatcaaaaag gatcttcacc 3961 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 4021 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 4081 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 4141 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 4201 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 4261 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 4321 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 4381 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 4441 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 4501 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 4561 agatgctttt ctgtgactgg tgaatactca accaagtcat tctgagaata gtgtatgcgg 4621 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 4681 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 4741 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 4801 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 4861 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 4921 atttatcagg gttattgtct catgaccaaa atcccttaac gtgagttttc gttccactga 4981 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 5041 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 5101 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 5161 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 5221 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 5281 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 5341 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 5401 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 5461 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 5521 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 5581 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 5641 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 5701 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 5761 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 5821 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 5881 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 5941 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 6001 tatgaccatg attacgccaa gctcagaatt aaccctcact aaagggacta gtcctgcagg 6061 tttaaacgaa ttcgcccttt gctttctctg accagcattc tctcccctgg gcctgtgccg 6121 ctttctgtct gcagcttgtg gcctgggtca cctctacggc tggcccagat ccttccctgc 6181 cgcctccttc aggttccgtc ttcctccact ccctcttccc cttgctctct gctgtgttgc 6241 tgcccaagga tgctctttcc ggagcacttc cttctcggcg ctgcaccacg tgatgtcctc 6301 tgagcggatc ctccccgtgt ctgggtcctc tccgggcatc tctcctccct cacccaaccc 6361 catgccgtct tcactcgctg ggttcccttt tccttctcct tctggggcct gtgccatctc 6421 tcgtttctta ggatggcctt ctccgacgga tgtctccctt gcgtcccgcc tccccttctt 6481 gtaggcctgc atcatcaccg tttttctgga caaccccaaa gtaccccgtc tccctggctt 6541 tagccacctc tccatcctct tgctttcttt gcctggacac cccgttctcc tgtggattcg 6601 ggtcacctct cactcctttc atttgggcag ctcccctacc ccccttacct ctctagtctg 6661 tgctagctct tccagccccc tgtcatggca tcttccaggg gtccgagagc tcagctagtc 6721 ttcttcctcc aacccgggcc cctatgtcca cttcaggaca gcatgtttgc tgcctccagg 6781 gatcctgtgt ccccgagctg ggaccacctt atattcccag ggccggttaa tgtggctctg 6841 gttctgggta cttttatctg tcccctccac cccacagtgg ggcaagcttc tgacctcttc 6901 tcttcctccc acagggcctc gagagttctg gcagcggaga gggcagagga agtcttctaa 6961 catgcggtga cgtggaggag aatcccggcc ctaggctcga gatgaccgag tacaagccca 7021 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt 7081 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca 7141 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg 7201 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg 7261 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc 7321 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca 7381 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg 7441 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca 7501 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag 7561 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg atctagaggg cccgtttaaa 7621 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 7681 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 7741 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg 7801 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta 7861 tgggtcaatt cgaacgctga cgtcatcaac ccgctccaag gaatcgcggg cccagtgtca 7921 ctaggcggga acacccagcg cgcgtgcgcc ctggcaggaa gatggctgtg agggacaggg 7981 gagtggcgcc ctgcaatatt tgcatgtcgc tatgtgttct gggaaatcac cataaacgtg 8041 aaatgtcttt ggatttggga atcttataag ttccctatca gtgatagaga tccctggtgc 8101 aggtggactg actcacctgc acctgtttta gagctagaaa tagcaagtta aaataaggct 8161 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgtcg acctcgacta 8221 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg 8281 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 8341 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 8401 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa 8461 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca 8521 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca 8581 tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc 8641 caattttgta tttatttatt ttttaattat tttgtgcagc ga //