LOCUS       KW734_pAAVS1-Nst        9751 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. Nst, 
            neomycin selection cassette. HA-R (delT2), without remaining CRISPR 
            protospacer and PAM.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9751)
  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: RDB13734
            Relation: alternative selection marker available: puromycin KW517 
            (RDB13735) 
            Remarks: After Gateway cloning, used in combination with AAVS1 
            nucleases: CRISPR/Cas9 KW516 (RDB13729), or TALEN KW226 (RDB13730) 
            and KW227 (RDB13731).
COMMENT     VNTDATE|763689600|
COMMENT     VNTDBDATE|763689600|
COMMENT     VNTNAME|KW734_pAAVS1-Nst-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             3127..3921
                     /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))"
     polyA_signal    3947..4171
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        4190..4569
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4571..4848
                     /label=chicken beta-actin promoter
     intron          4849..5864
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     protein_bind    5958..6082
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        6107..6137
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             6191..6850
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
     CDS             7192..7497
                     /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(7538..7662)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_site      7823..7878
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     misc_feature    8271..9090
                     /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(9108..9126)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9133..9149)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      9291..9746
                     /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 ggtaccatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     3181 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     3241 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     3301 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     3361 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     3421 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     3481 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     3541 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     3601 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     3661 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     3721 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     3781 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     3841 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     3901 cttcttgacg agttcttctg agtttaaacc cgctgatcag cctcgactgt gccttctagt
     3961 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     4021 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     4081 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     4141 aggcatgctg gggatgcggt gggctctatg ggctagcggt ggcggcctcg acattgatta
     4201 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     4261 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     4321 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     4381 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     4441 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     4501 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     4561 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc
     4621 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg
     4681 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg
     4741 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
     4801 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
     4861 cgctgccttc gccccgtgcc ccgctccgcg ccgcctcgcg ccgcccgccc cggctctgac
     4921 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
     4981 agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc
     5041 tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg
     5101 tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg
     5161 cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc
     5221 ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt
     5281 gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag
     5341 ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg
     5401 ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg
     5461 ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg
     5521 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt
     5581 tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc
     5641 gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt
     5701 cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct
     5761 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
     5821 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
     5881 tggttattgt gctgtctcat cattttggca aagaattcgc ggccgcggta ccggcgcgcc
     5941 ggatcctgca ggttatcaca agtttgtaca aaaaagctga acgagaaacg taaaatgata
     6001 taaatatcaa tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa
     6061 cacaacatat ccagtcacta tggcggccgc attaggcacc ccaggcttta cactttatgc
     6121 ttccggctcg tataatgtgt ggattttgag ttaggatccg tcgagatttt caggagctaa
     6181 ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca
     6241 tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt
     6301 tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc
     6361 ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattcc gtatggcaat
     6421 gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga
     6481 gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct
     6541 acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg
     6601 gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga
     6661 tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta
     6721 tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga
     6781 tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg
     6841 cggggcgtaa acgcgtggat ccggcttact aaaagccaga taacagtatg cgtatttgcg
     6901 cgctgatttt tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag
     6961 aggtatgcta tgaagcagcg tattacagtg acagttgaca gcgacagcta tcagttgctc
     7021 aaggcatata tgatgtcaat atctccggtc tggtaagcac aaccatgcag aatgaagccc
     7081 gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga agggatggct gaggtcgccc
     7141 ggtttattga aatgaacggc tcttttgctg acgagaacag gggctggtga aatgcagttt
     7201 aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat
     7261 attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca
     7321 gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg
     7381 atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc
     7441 agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg
     7501 tcaggctccc ttatacacag ccagtctgca ggtcgaccat agtgactgga tatgttgtgt
     7561 tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat
     7621 atcattttac gtttctcgtt cagctttctt gtacaaagtg gtgataactc tagagaattc
     7681 actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct
     7741 cacaaatacc actgagatct ttttccctct gccaaaaatt atggggacat catgaagccc
     7801 cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg
     7861 aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag
     7921 aatgagtatt tggtttagag tttggcaaca tatgccatat gctggctgcc atgaacaaag
     7981 gtggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca
     8041 tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc
     8101 tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg
     8161 actactccca gtcatagctg tccctcttct cttatgaaga tccctcgacc tgcagcccaa
     8221 gcttggatcc ctcgagttaa ttaaatctag aagtcgacag tactaagctt tgacagaaaa
     8281 gccccatcct taggcctcct ccttcctagt ctcctgatat tgggtctaac ccccacctcc
     8341 tgttaggcag attccttatc tggtgacaca cccccatttc ctggagccat ctctctcctt
     8401 gccagaacct ctaaggtttg cttacgatgg agccagagag gatcctggga gggagagctt
     8461 ggcagggggt gggagggaag ggggggatgc gtgacctgcc cggttctcag tggccaccct
     8521 gcgctaccct ctcccagaac ctgagctgct ctgacgcggc tgtctggtgc gtttcactga
     8581 tcctggtgct gcagcttcct tacacttccc aagaggagaa gcagtttgga aaaacaaaat
     8641 cagaataagt tggtcctgag ttctaacttt ggctcttcac ctttctagtc cccaatttat
     8701 attgttcctc cgtgcgtcag ttttacctgt gagataaggc cagtagccag ccccgtcctg
     8761 gcagggctgt ggtgaggagg ggggtgtccg tgtggaaaac tccctttgtg agaatggtgc
     8821 gtcctaggtg ttcaccaggt cgtggccgcc tctactccct ttctctttct ccatccttct
     8881 ttccttaaag agtccccagt gctatctggg acatattcct ccgcccagag cagggtcccg
     8941 cttccctaag gccctgctct gggcttctgg gtttgagtcc ttggcaagcc caggagaggc
     9001 gctcaggctt ccctgtcccc cttcctcgtc caccatctca tgcccctggc tctcctgccc
     9061 cttccctaca ggggttcctg gctctgctct agcgatcgcc aattcgccct atagtgagtc
     9121 gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     9181 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     9241 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg
     9301 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     9361 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     9421 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     9481 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     9541 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     9601 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     9661 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     9721 taacaaaata ttaacgctta caatttaggt g
//
