LOCUS       Exported                8624 bp ds-DNA     circular SYN 07-MAR-2016
DEFINITION  Donor vector targeting the human AAVS1 safe-harbor locus for 
            constitutive mCherry expression. P, puromycin selection cassette. 
            HA-R (delT2), without remaining CRISPR protospacer and PAM.
ACCESSION   .
VERSION     .
KEYWORDS    KW511_pAAVS1-P-CAG-mCh
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 8624)
  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 8624)
  AUTHORS   Knut Woltjen
  TITLE     Direct Submission
  JOURNAL   Exported Sunday, Jul 3, 2016 from SnapGene 3.1.4
            http://www.snapgene.com
COMMENT     ID: RDB13737
            Relation: alternative fluorescence available: eGFP KW317 (RDB13736) 
            Remarks: Used in combination with AAVS1 nucleases: CRISPR/Cas9 KW516
            (RDB13729), or TALEN KW226 (RDB13730) and KW227 (RDB13731).
FEATURES             Location/Qualifiers
     source          1..8624
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     promoter        21..125
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             126..986
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1157..1745
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2033..2054
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding site"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2069..2099
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    2107..2123
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /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
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2168..2186
                     /note="T3 promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    2199..3002
                     /note="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
                     /note="SA"
                     /note="splice acceptor site"
     CDS             3058..3111
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /note="T2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3121..3720
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /note="PuroR"
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    3758..3982
                     /note="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        4001..4380
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4382..4659
                     /note="chicken beta-actin promoter"
     intron          4660..5675
                     /note="chimeric intron"
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     protein_bind    5769..5793
                     /gene="mutant version of attB"
                     /bound_moiety="BP Clonase(TM)"
                     /note="attB1"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5799..6509
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /note="mCherry"
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     protein_bind    complement(6511..6535)
                     /gene="mutant version of attB"
                     /bound_moiety="BP Clonase(TM)"
                     /note="attB2"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_site      6696..6751
                     /note="beta-globin poly(A) signal"
                     /note="rabbit beta-globin polyadenylation signal"
     misc_feature    7144..7963
                     /note="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(7981..7999)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8006..8022)
                     /note="M13 fwd"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      8164..8619
                     /direction=RIGHT
                     /note="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 aaaaaagcag gctccaccat ggtgagcaag ggcgaggagg
     5821 acaacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga
     5881 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     5941 ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc
     6001 ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact
     6061 tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     6121 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga
     6181 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct
     6241 gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc
     6301 agaggctgaa gctgaaggac ggcggccact acgacgccga ggtcaagacc acctacaagg
     6361 ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagctg gacatcacct
     6421 cccacaacga ggactacacc atcgtggaac agtacgagcg cgccgagggc cgccactcca
     6481 ccggcggcat ggacgagctg tacaagtaag acccagcttt cttgtacaaa gtggtgataa
     6541 ctctagagaa ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc
     6601 caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga
     6661 catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc
     6721 aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca
     6781 tttaaaacat cagaatgagt atttggttta gagtttggca acatatgcca tatgctggct
     6841 gccatgaaca aaggtggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc
     6901 attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata ttttgttttg
     6961 tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt
     7021 tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcttatga agatccctcg
     7081 acctgcagcc caagcttgga tccctcgagt taattaaatc tagaagtcga cagtactaag
     7141 ctttgacaga aaagccccat ccttaggcct cctccttcct agtctcctga tattgggtct
     7201 aacccccacc tcctgttagg cagattcctt atctggtgac acacccccat ttcctggagc
     7261 catctctctc cttgccagaa cctctaaggt ttgcttacga tggagccaga gaggatcctg
     7321 ggagggagag cttggcaggg ggtgggaggg aaggggggga tgcgtgacct gcccggttct
     7381 cagtggccac cctgcgctac cctctcccag aacctgagct gctctgacgc ggctgtctgg
     7441 tgcgtttcac tgatcctggt gctgcagctt ccttacactt cccaagagga gaagcagttt
     7501 ggaaaaacaa aatcagaata agttggtcct gagttctaac tttggctctt cacctttcta
     7561 gtccccaatt tatattgttc ctccgtgcgt cagttttacc tgtgagataa ggccagtagc
     7621 cagccccgtc ctggcagggc tgtggtgagg aggggggtgt ccgtgtggaa aactcccttt
     7681 gtgagaatgg tgcgtcctag gtgttcacca ggtcgtggcc gcctctactc cctttctctt
     7741 tctccatcct tctttcctta aagagtcccc agtgctatct gggacatatt cctccgccca
     7801 gagcagggtc ccgcttccct aaggccctgc tctgggcttc tgggtttgag tccttggcaa
     7861 gcccaggaga ggcgctcagg cttccctgtc ccccttcctc gtccaccatc tcatgcccct
     7921 ggctctcctg ccccttccct acaggggttc ctggctctgc tctagcgatc gccaattcgc
     7981 cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     8041 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     8101 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     8161 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     8221 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     8281 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     8341 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     8401 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     8461 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     8521 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     8581 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtg
//
