LOCUS Exported 8022 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION For targeting shRNA construct into human AAVS1 locus, using genome editing. Expresses shRNA of interest (cloning: EcoRI and AgeI) under U6 promoter, flanked by AAVS1 homology arms.. ACCESSION . VERSION . KEYWORDS pAAVS1-shRNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8022) AUTHORS Daneshvar K, Pondick JV, Kim BM, Zhou C, York SR, Macklin JA, Abualteen A, Tan B, Sigova AA, Marcho C, Tremblay KD, Mager J, Choi MY, Mullen AC TITLE DIGIT Is a Conserved Long Noncoding RNA that Regulates GSC Expression to Control Definitive Endoderm Differentiation of Embryonic Stem Cells. JOURNAL Cell Rep. 2016 Oct 4;17(2):353-365. doi: 10.1016/j.celrep.2016.09.017. PUBMED 27705785 REFERENCE 2 (bases 1 to 8022) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8022 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 491..510 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(729..833) /gene="bla" /label=AmpR promoter primer_bind 901..919 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(957..979) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 1079..1098 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 1292..1314 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 1306..1323 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 1307..1323 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" misc_feature 1325..2128 /label=HA-L /note="left homology arm from the adeno-associated virus integration site (AAVS1) within intron 1 of the human PPP1R12C gene" promoter 2158..2398 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2158..2178 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2329..2348 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_feature 4320..4437 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 4497..4997 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 4916..4934 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 5019..5618 /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(5019..5038) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5515..5535 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 5625..5849 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" misc_feature 5863..6699 /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(6729..6745) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6729..6745) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6742..6764) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6753..6769 /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(6777..6807) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6822..6843 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6960..6977) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7131..7719) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7211..7230) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7890..728) /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" ORIGIN 1 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 61 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 121 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 181 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 241 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 301 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 361 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 421 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 481 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 541 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 601 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 661 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 721 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 781 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 841 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 901 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc 961 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga 1021 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg 1081 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc 1141 gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa 1201 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca 1261 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc 1321 agtgtgcttt ctctgaccag cattctctcc cctgggcctg tgccgctttc tgtctgcagc 1381 ttgtggcctg ggtcacctct acggctggcc cagatccttc cctgccgcct ccttcaggtt 1441 ccgtcttcct ccactccctc ttccccttgc tctctgctgt gttgctgccc aaggatgctc 1501 tttccggagc acttccttct cggcgctgca ccacgtgatg tcctctgagc ggatcctccc 1561 cgtgtctggg tcctctccgg gcatctctcc tccctcaccc aaccccatgc cgtcttcact 1621 cgctgggttc ccttttcctt ctccttctgg ggcctgtgcc atctctcgtt tcttaggatg 1681 gccttctccg acggatgtct cccttgcgtc ccgcctcccc ttcttgtagg cctgcatcat 1741 caccgttttt ctggacaacc ccaaagtacc ccgtctccct ggctttagcc acctctccat 1801 cctcttgctt tctttgcctg gacaccccgt tctcctgtgg attcgggtca cctctcactc 1861 ctttcatttg ggcagctccc ctacccccct tacctctcta gtctgtgcta gctcttccag 1921 ccccctgtca tggcatcttc caggggtccg agagctcagc tagtcttctt cctccaaccc 1981 gggcccctat gtccacttca ggacagcatg tttgctgcct ccagggatcc tgtgtccccg 2041 agctgggacc accttatatt cccagggccg gttaatgtgg ctctggttct gggtactttt 2101 atctgtcccc tccaccccac agtggggcac tagcctcgag cggccgcccc cttcaccgag 2161 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata 2221 attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag 2281 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg 2341 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga 2401 aacaccggtg tgttgtaaat gagcacacaa aatacacatg ctaaaatatt atattctatg 2461 acctttataa aatcaaccaa aatcttcttt ttaataactt tagtatcaat aattagaatt 2521 tttatgttcc tttttgcaaa cttttaataa aaatgagcaa aataaaaaaa cgctagtttt 2581 agtaactcgc gttgttttct tcacctttaa taatagctac tccaccactt gttcctaagc 2641 ggtcagctcc tgcttcaatc attttttgag catcttcaaa tgttctaact ccaccagctg 2701 ctttaactaa agcattgtct ttaacaactg acttcattag tttaacatct tcaaatgttg 2761 cacctgattt tgaaaatcct gttgatgttt taacaaattc taatccagct tcaacagcta 2821 tttcacaagc tttcatgatt tcttcttttg ttaataaaca attttccata atacatttaa 2881 caacatgtga tccagctgct ttttttacag ctttcatgtc ttctaaaact aattcataat 2941 ttttgtcttt taatgcacca atatttaata ccatatcaat ttctgttgca ccatctttaa 3001 ttgcttcaga aacttcgaat gcttttgtag ctgttgtgca tgcacctaga ggaaaaccta 3061 caacatttgt tattcctaca tttgtgcctt ttaataattc tttacaatag cttgttcaat 3121 atgaattaac acaaactgtt gcaaaatcaa attcaattgc ttcatcacat aattgtttaa 3181 tttcagcttt cgtagcatct tgttttaata atgtgtgatc tatatatttg tttagtttca 3241 ttttttctcc tatatattca tttttaattt taattcttta ataatttcgt ctactttaac 3301 tttagcgttt tgaacagatt caccaacacc tataaaataa atttttagtt taggttcagt 3361 tccacttggg cgaacagcaa atcatgactt atcttctaaa taaaatttta gtaagtcttg 3421 tcctggcata ttatacattc catcgatgta gtcttcaaca ttaacaactt taagtccagc 3481 aatttgagtt aagggtgttg ctctcaatga tttcattaat ggttcaattt ttaatttctt 3541 ttcttctggt ttaaaattca agtttaaagt gaaagtgtaa tatgcaccca tttctttaaa 3601 taaatcttct aaatagtcta ctaatgtttt attttgtttt ttataaaatc aagcagcctc 3661 tgctattaat atagaagctt gtattccatc tttatctcta gctgagtcat caattacata 3721 tccataactt tcttcataag caaaaacaaa atttaatccg ttatcttctt ctttagcaat 3781 ttctctaccc attcatttaa atccagttaa agtttttaca atattaactc catatttttc 3841 atgagcgatt ctatcaccca aatcacttgt tacaaaactt gaatatagag ccggattttt 3901 tggaatgcta tttaagcgtt ttagatttga taattttcaa tcaattaaaa ttggtcctgt 3961 ttgatttcca tctaatctta caaaatgacc atcatgtttt attgccattc caaatctgtc 4021 agcatctggg tcattcataa taataatatc tgcatcatgt ttaataccat attcaagcgg 4081 tatttttcat gcaggatcaa attctggatt tggatttaca acatttttaa atgtttcatc 4141 ttcaaatgca tgctcttcaa cctcaataac gttatatcct gattcacgta atatttttgg 4201 ggtaaattta gttcctgttc cattaactgc gctaaaaata atttttaaat cttttttagc 4261 ttcttgctct tttttgtaga attctcgacc tcgagacaaa tggcagtatt catccacaat 4321 tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata 4381 gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg 4441 gtttattaca gggacagcag agatccactt tggccgcggc tcgaaagctt ttggggttgc 4501 gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg cgtggttccg 4561 ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt cgcagcgtca 4621 cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa 4681 gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc 4741 tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat 4801 gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc cgggaagggg 4861 cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt 4921 ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca 4981 ccgacctctc tccccagggg gatccaccgg agcttaccat gaccgagtac aagcccacgg 5041 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg 5101 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg 5161 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg 5221 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt 5281 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac 5341 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg 5401 tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag 5461 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc 5521 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac 5581 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgagg taccctgtgc cttctagttg 5641 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc 5701 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 5761 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 5821 gcatgctggg gatgcggtgg gctctatggg gtaccaagct ttactaggga caggattggt 5881 gacagaaaag ccccatcctt aggcctcctc cttcctagtc tcctgatatt gggtctaacc 5941 cccacctcct gttaggcaga ttccttatct ggtgacacac ccccatttcc tggagccatc 6001 tctctccttg ccagaacctc taaggtttgc ttacgatgga gccagagagg atcctgggag 6061 ggagagcttg gcagggggtg ggagggaagg gggggatgcg tgacctgccc ggttctcagt 6121 ggccaccctg cgctaccctc tcccagaacc tgagctgctc tgacgcggct gtctggtgcg 6181 tttcactgat cctggtgctg cagcttcctt acacttccca agaggagaag cagtttggaa 6241 aaacaaaatc agaataagtt ggtcctgagt tctaactttg gctcttcacc tttctagtcc 6301 ccaatttata ttgttcctcc gtgcgtcagt tttacctgtg agataaggcc agtagccagc 6361 cccgtcctgg cagggctgtg gtgaggaggg gggtgtccgt gtggaaaact ccctttgtga 6421 gaatggtgcg tcctaggtgt tcaccaggtc gtggccgcct ctactccctt tctctttctc 6481 catccttctt tccttaaaga gtccccagtg ctatctggga catattcctc cgcccagagc 6541 agggtcccgc ttccctaagg ccctgctctg ggcttctggg tttgagtcct tggcaagccc 6601 aggagaggcg ctcaggcttc cctgtccccc ttcctcgtcc accatctcat gcccctggct 6661 ctcctgcccc ttccctacag gggttcctgg ctctgctctt gcaggcatgc aagcttggcg 6721 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 6781 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 6841 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 6901 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 6961 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 7021 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 7081 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 7141 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 7201 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 7261 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 7321 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 7381 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 7441 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 7501 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 7561 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 7621 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 7681 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 7741 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 7801 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 7861 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 7921 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 7981 acgatacggg agggcttacc atctggcccc agtgctgcaa tg //