LOCUS       sequence-174872-        7417 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV backbone plasmid including luc-pA knock-in donor and Ai14gRNA 
            for HITI .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7417)
  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 7417)
  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-luc
FEATURES             Location/Qualifiers
     source          1..7417
                     /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             596..2245
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     polyA_signal    2300..2421
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2311..2330)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2365..2384
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2609..2820
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2833..3101
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      3125..3134
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3131..3133,3134..3136,3137..3847)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3131..3133,3134..3136,3137..3847)
                     /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(3176..3197)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3437..3456)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3784..3805
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4164..4640
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4363..4382)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4680..4820
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4680..4809
                     /label=AAV2 ITR
     rep_origin      4895..5350
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4982..5001)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5192..5213
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5367..5386)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5486..5508
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5546..5564)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5632..5736
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5737..5805,5806..6597)
                     /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(5955..5974)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6768..7356
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7257..7276
                     /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 ccaccatgga
      601 agatgccaaa aacattaaga agggcccagc gccattctac ccactcgaag acgggaccgc
      661 cggcgagcag ctgcacaaag ccatgaagcg ctacgccctg gtgcccggca ccatcgcctt
      721 taccgacgca catatcgagg tggacattac ctacgccgag tacttcgaga tgagcgttcg
      781 gctggcagaa gctatgaagc gctatgggct gaatacaaac catcggatcg tggtgtgcag
      841 cgagaatagc ttgcagttct tcatgcccgt gttgggtgcc ctgttcatcg gtgtggctgt
      901 ggccccagct aacgacatct acaacgagcg cgagctgctg aacagcatgg gcatcagcca
      961 gcccaccgtc gtattcgtga gcaagaaagg gctgcaaaag atcctcaacg tgcaaaagaa
     1021 gctaccgatc atacaaaaga tcatcatcat ggatagcaag accgactacc agggcttcca
     1081 aagcatgtac accttcgtga cttcccattt gccacccggc ttcaacgagt acgacttcgt
     1141 gcccgagagc ttcgaccggg acaaaaccat cgccctgatc atgaacagta gtggcagtac
     1201 cggattgccc aagggcgtag ccctaccgca ccgcaccgct tgtgtccgat tcagtcatgc
     1261 ccgcgacccc atcttcggca accagatcat ccccgacacc gctatcctca gcgtggtgcc
     1321 atttcaccac ggcttcggca tgttcaccac gctgggctac ttgatctgcg gctttcgggt
     1381 cgtgctcatg taccgcttcg aggaggagct attcttgcgc agcttgcaag actataagat
     1441 tcaatctgcc ctgctggtgc ccacactatt tagcttcttc gctaagagca ctctcatcga
     1501 caagtacgac ctaagcaact tgcacgagat cgccagcggc ggggcgccgc tcagcaagga
     1561 ggtaggtgag gccgtggcca aacgcttcca cctaccaggc atccgccagg gctacggcct
     1621 gacagaaaca accagcgcca ttctgatcac ccccgaaggg gacgacaagc ctggcgcagt
     1681 aggcaaggtg gtgcccttct tcgaggctaa ggtggtggac ttggacaccg gtaagacact
     1741 gggtgtgaac cagcgcggcg agctgtgcgt ccgtggcccc atgatcatga gcggctacgt
     1801 taacaacccc gaggctacaa acgctctcat cgacaaggac ggctggctgc acagcggcga
     1861 catcgcctac tgggacgagg acgagcactt cttcatcgtg gaccggctga agagcctgat
     1921 caaatacaag ggctaccagg tagccccagc cgaactggag agcatcctgc tgcaacaccc
     1981 caacatcttc gacgccgggg tcgccggcct gcccgacgac gatgccggcg agctgcccgc
     2041 cgcagtcgtc gtgctggaac acggtaaaac catgaccgag aaggagatcg tggactatgt
     2101 ggccagccag gttacaaccg ccaagaagct gcgcggtggt gttgtgttcg tggacgaggt
     2161 gcctaaagga ctgaccggca agttggacgc ccgcaagatc cgcgagattc tcattaaggc
     2221 caagaagggc ggcaagatcg ccgtgaattc ttaactgcag tctagagtcg gggcggccgg
     2281 ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     2341 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     2401 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
     2461 ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatcgat
     2521 aaggatccgt cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccgaggaac
     2581 ttcttagggc ccgcggacgc gtgtcagtgg gcagagcgca catcgcccac agtccccgag
     2641 aagttggggg gaggggtcgg caattgaacg ggtgcctaga gaaggtggcg cggggtaaac
     2701 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     2761 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     2821 ctgaagcttc gaggggctcg catctctcct tcacgcgccc gccgccctac ctgaggccgc
     2881 catccacgcc ggttgagtcg cgttctgccg cctcccgcct gtggtgcctc ctgaactgcg
     2941 tccgccgtct aggtaagttt aaagctcagg tcgagaccgg gcctttgtcc ggcgctccct
     3001 tggagcctac ctagactcag ccggctctcc acgctttgcc tgaccctgct tgctcaactc
     3061 tacgtctttg tttcgttttc tgttctgcgc cgttacagat ccaagctgtg accggcgcgt
     3121 cgacgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     3181 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     3241 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     3301 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     3361 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     3421 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     3481 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     3541 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     3601 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     3661 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     3721 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     3781 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     3841 gtacaagtcc ggactcagat ctcgagagga ggaggaggag acagacagca ggatgcccca
     3901 cctcgacagc cccggcagct cccagccgag acgctccttc ctctcaaggg tgatcagggc
     3961 agcgctaccg ttgcagctgc ttctgctgct gctgctgctc ctggcctgcc tgctacctgc
     4021 ctctgaagat gactacagct gcacccaggc caacaacttt gcccgatcct tctaccccat
     4081 gctgcggtac accaacgggc cacctcccac ctaggaattc gatatcaagc ttatcgatac
     4141 cgagcgctgc tcgagagatc tacgggtggc atccctgtga cccctcccca gtgcctctcc
     4201 tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa ttaagttgca
     4261 tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga
     4321 gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg gaaccaagct
     4381 ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt caagcgattc
     4441 tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg ctcagctaat
     4501 ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc tccaactcct
     4561 aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca
     4621 ctgctccctt ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg agcggccgca
     4681 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     4741 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     4801 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4861 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     4921 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc
     4981 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     5041 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     5101 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     5161 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     5221 ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat
     5281 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     5341 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     5401 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     5461 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     5521 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     5581 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     5641 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5701 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5761 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5821 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5881 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5941 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     6001 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     6061 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     6121 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     6181 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6241 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6301 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6361 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6421 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6481 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6541 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6601 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6661 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6721 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6781 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6841 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6901 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6961 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     7021 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     7081 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     7141 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     7201 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     7261 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7321 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7381 cggttcctgg ccttttgctg gccttttgct cacatgt
//
