LOCUS       sequence-174241-        6937 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV backbone plasmid including GFP knock-in donor and Tubb3gRNA for 
            HITI .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6937)
  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 6937)
  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-mTubb3
FEATURES             Location/Qualifiers
     source          1..6937
                     /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"
     CDS             join(591..593,594..1304)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(591..593,594..1304)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(633..654)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(894..913)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1241..1262
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        1421..2599
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1652..2590
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2547..2567
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2624..2633
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2636..2662
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2663..3367
                     /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(2805..2823)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3073..3092
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3684..4160
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3883..3902)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4200..4340
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4200..4329
                     /label=AAV2 ITR
     rep_origin      4415..4870
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4502..4521)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4712..4733
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4887..4906)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5006..5028
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5066..5084)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5152..5256
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5257..5325,5326..6117)
                     /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(5475..5494)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6288..6876
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6777..6796
                     /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 gaaacaccgg ctgcgagcaa
      421 cttcacttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
      481 gaaaaagtgg caccgagtcg gtgctttttt tctagaccca gctttcttgt acaaagttgg
      541 cgtttaaacg gctgcgagca acttcacttg ggtatgccgg cggtagcgct gtgagcaagg
      601 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
      661 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
      721 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
      781 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
      841 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
      901 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
      961 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1021 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     1081 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1141 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1201 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1261 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtggctg cgagcaactt
     1321 cacttgggac gcgtaagctt tgcaaagatg gataaagttt taaacagaga ggaatctttg
     1381 cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg
     1441 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
     1501 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
     1561 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
     1621 ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc
     1681 tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta
     1741 cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc
     1801 ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg
     1861 cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat
     1921 ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc
     1981 gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg
     2041 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg
     2101 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat
     2161 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg
     2221 gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg
     2281 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg
     2341 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag
     2401 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg
     2461 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct
     2521 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct
     2581 tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gacgccacca tggcatatcc
     2641 gtatgatgtg ccggattatg cggtgagcaa gggcgaggag gataacatgg ccatcatcaa
     2701 ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat
     2761 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac
     2821 caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc
     2881 caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga
     2941 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca
     3001 ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt
     3061 cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg
     3121 gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga
     3181 cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct
     3241 gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac
     3301 catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct
     3361 gtacaagtcc ggactcagat ctcgagagga ggaggaggag acagacagca ggatgcccca
     3421 cctcgacagc cccggcagct cccagccgag acgctccttc ctctcaaggg tgatcagggc
     3481 agcgctaccg ttgcagctgc ttctgctgct gctgctgctc ctggcctgcc tgctacctgc
     3541 ctctgaagat gactacagct gcacccaggc caacaacttt gcccgatcct tctaccccat
     3601 gctgcggtac accaacgggc cacctcccac ctaggaattc gatatcaagc ttatcgatac
     3661 cgagcgctgc tcgagagatc tacgggtggc atccctgtga cccctcccca gtgcctctcc
     3721 tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa ttaagttgca
     3781 tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga
     3841 gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg gaaccaagct
     3901 ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt caagcgattc
     3961 tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg ctcagctaat
     4021 ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc tccaactcct
     4081 aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca
     4141 ctgctccctt ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg agcggccgca
     4201 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     4261 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     4321 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4381 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     4441 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc
     4501 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     4561 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4621 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     4681 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4741 ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat
     4801 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4861 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4921 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4981 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     5041 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     5101 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     5161 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5221 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5281 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5341 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5401 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5461 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5521 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5581 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5641 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5701 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5761 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5821 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5881 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     5941 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6001 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6061 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6121 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6181 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6241 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6301 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6361 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6421 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6481 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6541 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6601 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6661 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6721 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6781 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6841 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6901 cggttcctgg ccttttgctg gccttttgct cacatgt
//
