LOCUS       sequence-174873-        7136 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 7136)
  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 7136)
  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..7136
                     /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..3286
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2339..3277
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3234..3254
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3311..3320
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3323..3349
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3350..4054
                     /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(3492..3510)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3760..3779
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4328..4376
                     /note="synthetic polyadenylation signal"
     repeat_region   4399..4539
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4399..4528
                     /label=AAV2 ITR
     rep_origin      4614..5069
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4701..4720)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4911..4932
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5086..5105)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5205..5227
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5265..5283)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5351..5455
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5456..5524,5525..6316)
                     /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(5674..5693)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6487..7075
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6976..6995
                     /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 tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag
     2461 tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga
     2521 ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct
     2581 cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt
     2641 tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt
     2701 tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg
     2761 cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct
     2821 ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc
     2881 ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa
     2941 atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc
     3001 ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca
     3061 cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta
     3121 tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt
     3181 gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc
     3241 tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgaggta ccggatcctc
     3301 tagagtcgac gccaccatgg catatccgta tgatgtgccg gattatgcgg tgagcaaggg
     3361 cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg
     3421 ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg
     3481 cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat
     3541 cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc
     3601 cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga
     3661 ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta
     3721 caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac
     3781 catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga
     3841 gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac
     3901 ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga
     3961 catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg
     4021 ccactccacc ggcggcatgg acgagctgta caagtccgga ctcagatctc gagaggagga
     4081 ggaggagaca gacagcagga tgccccacct cgacagcccc ggcagctccc agccgagacg
     4141 ctccttcctc tcaagggtga tcagggcagc gctaccgttg cagctgcttc tgctgctgct
     4201 gctgctcctg gcctgcctgc tacctgcctc tgaagatgac tacagctgca cccaggccaa
     4261 caactttgcc cgatccttct accccatgct gcggtacacc aacgggccac ctcccaccta
     4321 ggaattcaat aaaagatctt tattttcatt agatctgtgt gttggttttt tgtgtgcacg
     4381 tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     4441 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     4501 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     4561 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     4621 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4681 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4741 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4801 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     4861 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4921 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
     4981 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     5041 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     5101 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     5161 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     5221 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     5281 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     5341 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5401 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5461 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5521 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5581 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5641 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5701 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5761 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5821 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5881 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5941 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6001 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6061 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6121 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     6181 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6241 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6301 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     6361 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6421 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6481 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6541 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6601 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6661 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6721 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6781 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6841 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6901 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6961 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     7021 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     7081 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
