LOCUS       sequence-169996-        7135 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV backbone plasmid including GFPNLS-pA knock-in donor and Ai14gRNA
            for HITI .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7135)
  AUTHORS   Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, 
            Hatanaka F, Yamamoto M, Araoka T, Li Z, Kurita M, Hishida T, Li M, 
            Aizawa E, Guo S, Chen S, Goebl A, Soligalla RD, Qu J, Jiang T, Fu X,
            Jafari M, Esteban CR, Berggren WT, Lajara J, Nunez-Delicado E, 
            Guillen P, Campistol JM, Matsuzaki F, Liu GH, Magistretti P, Zhang 
            K, Callaway EM, Zhang K, Belmonte JC
  TITLE     In vivo genome editing via CRISPR/Cas9 mediated homology-independent
            targeted integration.
  JOURNAL   Nature. 2016 Dec 1;540(7631):144-149. doi: 10.1038/nature20565. Epub
            2016 Nov 16.
  PUBMED    27851729
REFERENCE   2  (bases 1 to 7135)
  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: 
            "10.1038/nature20565"; journalName: "Nature"; date: "2016-12-1- 1"; 
            volume: "540"; issue: "7631"; pages: "144-149"
COMMENT     Sequence Label: pAAV-Ai14-HITI
FEATURES             Location/Qualifiers
     source          1..7135
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        160..408
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     160..180
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     331..350
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        429..504
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(516..540)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      590..599
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(596..598,599..601,602..1312)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(596..598,599..601,602..1312)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(641..662)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(902..921)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1249..1270
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1331..1351
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1355..1375
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1379..1399
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1473..1492)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1538..1593
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1592..1611)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     promoter        2108..3285
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2339..3276
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3233..3253
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3310..3319
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3322..3348
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3349..4053
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(3491..3509)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3759..3778
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4327..4375
                     /note="synthetic polyadenylation signal"
     repeat_region   4398..4538
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4398..4527
                     /label=AAV2 ITR
     rep_origin      4613..5068
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4700..4719)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4910..4931
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5085..5104)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5204..5226
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5264..5282)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5350..5454
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5455..5523,5524..6315)
                     /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"
     primer_bind     complement(5673..5692)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6486..7074
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6975..6994
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaaggtc gggcaggaag agggcctatt tcccatgatt
      181 ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat taatttgact
      241 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
      301 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
      361 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga ggaacttctt
      421 agggcccggt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
      481 gaaaaagtgg caccgagtcg gtgctttttt tctagaccca gctttcttgt acaaagttgg
      541 cgtttaaacc cccgggcgcg actatagagg aacttcttag ggcccgcggg ccaccatggt
      601 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
      661 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
      721 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
      781 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
      841 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
      901 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
      961 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1021 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     1081 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     1141 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1201 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1261 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtccggact
     1321 cagatctgat ccaaaaaaga agagaaaggt agatccaaaa aagaagagaa aggtagatcc
     1381 aaaaaagaag agaaaggtat aaaagcttcg aggaattcga agcttctagg taccagatat
     1441 caggatcctg gccagctagc taggtagcta gatctttttc cctctgccaa aaattatggg
     1501 gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt
     1561 gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat
     1621 catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg
     1681 gctgccatga acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct
     1741 gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt
     1801 tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga
     1861 tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc
     1921 ctcgaccgat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     1981 tgggcgctct tcgaggaact tcttagggcc cgcggacgcg taagctttgc aaagatggat
     2041 aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct tgaaaggagt
     2101 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
     2161 gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg
     2221 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
     2281 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt
     2341 aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc
     2401 ttgaattact tccactggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt
     2461 gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag
     2521 gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc
     2581 gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt
     2641 ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt
     2701 ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc
     2761 ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg
     2821 gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg
     2881 gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa
     2941 tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc
     3001 tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac
     3061 ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat
     3121 gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg
     3181 atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct
     3241 cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgaggtac cggatcctct
     3301 agagtcgacg ccaccatggc atatccgtat gatgtgccgg attatgcggt gagcaagggc
     3361 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
     3421 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     3481 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     3541 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     3601 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     3661 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     3721 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
     3781 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     3841 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     3901 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     3961 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     4021 cactccaccg gcggcatgga cgagctgtac aagtccggac tcagatctcg agaggaggag
     4081 gaggagacag acagcaggat gccccacctc gacagccccg gcagctccca gccgagacgc
     4141 tccttcctct caagggtgat cagggcagcg ctaccgttgc agctgcttct gctgctgctg
     4201 ctgctcctgg cctgcctgct acctgcctct gaagatgact acagctgcac ccaggccaac
     4261 aactttgccc gatccttcta ccccatgctg cggtacacca acgggccacc tcccacctag
     4321 gaattcaata aaagatcttt attttcatta gatctgtgtg ttggtttttt gtgtgcacgt
     4381 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4441 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     4501 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     4561 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     4621 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4681 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4741 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4801 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     4861 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4921 ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt
     4981 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5041 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     5101 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     5161 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     5221 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     5281 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     5341 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5401 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5461 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5521 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5581 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5641 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5701 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5761 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5821 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5881 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5941 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6001 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6061 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6121 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6181 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6241 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6301 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6361 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6421 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6481 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6541 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6601 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     6661 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6721 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6781 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6841 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6901 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6961 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7021 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7081 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
