LOCUS       sequence-157348-        8022 bp    DNA     circular SYN 15-DEC-2025
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    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2016.09"; journalName: "Cell Rep"; date: 
            "2016-10-4- 4"; volume: "17"; issue: "2"; pages: "353-365"
COMMENT     Sequence Label: pAAVS1-shRNA
FEATURES             Location/Qualifiers
     source          1..8022
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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..2406
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     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        4487..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(join(6777..6783,6784..6801,6802..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(join(7890..8022,1..659,660..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
//
