LOCUS       KW517_pAAVS1-P-C        9562 bp ds-DNA     circular SYN 07-MAR-2016
DEFINITION  Donor vector targeting the human AAVS1 safe-harbor locus compatable 
            with Gateway cloning of cDNAs for constitutive expression. P, puro 
            selection cassette. HA-R (delT2), without remaining CRISPR 
            protospacer and PAM.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9562)
  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.
  PUBMED    26707206
REFERENCE   2  (bases 1 to 9562)
  AUTHORS   Knut Woltjen
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Apr 1, 2016 from SnapGene 3.1.2 to Vector NTI(R) 
            format
            http://www.snapgene.com
COMMENT     ID: RDB13735
            Relation: alternative selection marker available: neomycin KW734 
            (RDB13734) 
            Remarks: After Gateway cloning, used in combination with AAVS1 
            nucleases: CRISPR/Cas9 KW516 (RDB13729), or TALEN KW226 (RDB13730) 
            and KW227 (RDB13731).
COMMENT     VNTDATE|753321600|
COMMENT     VNTDBDATE|763689600|
COMMENT     VNTNAME|KW517_pAAVS1-P-CAG-DEST|
COMMENT     VNTAUTHORNAME|Knut Woltjen|
FEATURES             Location/Qualifiers
     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"
     rep_origin      1157..1745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     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     2131..2147
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     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."
     CDS             3121..3720
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
     polyA_signal    3758..3982
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        4001..4380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4382..4659
                     /label=chicken beta-actin promoter
     intron          4660..5675
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     protein_bind    5769..5893
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        5918..5948
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             6002..6661
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
     CDS             7003..7308
                     /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."
     protein_bind    complement(7349..7473)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_site      7634..7689
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     misc_feature    8082..8901
                     /label=HA-R (delT2)
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene; AAVS1 T2 sgRNA protospacer and PAM sequences
                     removed (ACTAGGGACAGGATTGG)"
     promoter        complement(8919..8937)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8944..8960)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      9102..9557
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
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 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     3181 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     3241 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     3301 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     3361 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     3421 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     3481 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     3541 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     3601 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     3661 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     3721 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     3781 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     3841 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     3901 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     3961 ggggatgcgg tgggctctat gggctagcgg tggcggcctc gacattgatt attgactagt
     4021 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     4081 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     4141 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     4201 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     4261 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     4321 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     4381 gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca
     4441 attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg
     4501 gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag gcggagaggt
     4561 gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg
     4621 cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt
     4681 cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
     4741 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagcgcttgg
     4801 tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg
     4861 gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg
     4921 ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc gcggggcttt
     4981 gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg cggtgccccg cggtgcgggg
     5041 ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg tgagcagggg
     5101 gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga gttgctgagc
     5161 acggcccggc ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc gccgtgccgg
     5221 gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg gccggggagg
     5281 gctcggggga ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc
     5341 gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct ttgtcccaaa
     5401 tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcggggcga
     5461 agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc
     5521 gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc tgccttcggg
     5581 ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg
     5641 ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg
     5701 tgctgtctca tcattttggc aaagaattcg cggccgcggt accggcgcgc cggatcctgc
     5761 aggttatcac aagtttgtac aaaaaagctg aacgagaaac gtaaaatgat ataaatatca
     5821 atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa acacaacata
     5881 tccagtcact atggcggccg cattaggcac cccaggcttt acactttatg cttccggctc
     5941 gtataatgtg tggattttga gttaggatcc gtcgagattt tcaggagcta aggaagctaa
     6001 aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga
     6061 acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga
     6121 tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat
     6181 tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa tgaaagacgg
     6241 tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga
     6301 aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata
     6361 ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga
     6421 gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt
     6481 ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg
     6541 cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg atggcttcca
     6601 tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta
     6661 aacgcgtgga tccggcttac taaaagccag ataacagtat gcgtatttgc gcgctgattt
     6721 ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaaa gaggtatgct
     6781 atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat
     6841 atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc
     6901 gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg
     6961 aaatgaacgg ctcttttgct gacgagaaca ggggctggtg aaatgcagtt taaggtttac
     7021 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
     7081 acgcccgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
     7141 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
     7201 gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc
     7261 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggctcc
     7321 cttatacaca gccagtctgc aggtcgacca tagtgactgg atatgttgtg ttttacagta
     7381 ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta tatcatttta
     7441 cgtttctcgt tcagctttct tgtacaaagt ggtgataact ctagagaatt cactcctcag
     7501 gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac
     7561 cactgagatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat
     7621 ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg
     7681 tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat
     7741 ttggtttaga gtttggcaac atatgccata tgctggctgc catgaacaaa ggtggctata
     7801 aagaggtcat cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc
     7861 cttgacttga ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc
     7921 cctaaaattt tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc
     7981 agtcatagct gtccctcttc tcttatgaag atccctcgac ctgcagccca agcttggatc
     8041 cctcgagtta attaaatcta gaagtcgaca gtactaagct ttgacagaaa agccccatcc
     8101 ttaggcctcc tccttcctag tctcctgata ttgggtctaa cccccacctc ctgttaggca
     8161 gattccttat ctggtgacac acccccattt cctggagcca tctctctcct tgccagaacc
     8221 tctaaggttt gcttacgatg gagccagaga ggatcctggg agggagagct tggcaggggg
     8281 tgggagggaa gggggggatg cgtgacctgc ccggttctca gtggccaccc tgcgctaccc
     8341 tctcccagaa cctgagctgc tctgacgcgg ctgtctggtg cgtttcactg atcctggtgc
     8401 tgcagcttcc ttacacttcc caagaggaga agcagtttgg aaaaacaaaa tcagaataag
     8461 ttggtcctga gttctaactt tggctcttca cctttctagt ccccaattta tattgttcct
     8521 ccgtgcgtca gttttacctg tgagataagg ccagtagcca gccccgtcct ggcagggctg
     8581 tggtgaggag gggggtgtcc gtgtggaaaa ctccctttgt gagaatggtg cgtcctaggt
     8641 gttcaccagg tcgtggccgc ctctactccc tttctctttc tccatccttc tttccttaaa
     8701 gagtccccag tgctatctgg gacatattcc tccgcccaga gcagggtccc gcttccctaa
     8761 ggccctgctc tgggcttctg ggtttgagtc cttggcaagc ccaggagagg cgctcaggct
     8821 tccctgtccc ccttcctcgt ccaccatctc atgcccctgg ctctcctgcc ccttccctac
     8881 aggggttcct ggctctgctc tagcgatcgc caattcgccc tatagtgagt cgtattacaa
     8941 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     9001 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     9061 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     9121 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     9181 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     9241 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     9301 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     9361 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     9421 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     9481 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     9541 attaacgctt acaatttagg tg
//
