LOCUS       sequence-417139-        4694 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 promoter expressing sgClta. Clta homology arms, sfGFP knock-in..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4694)
  AUTHORS   Nyberg WA, Ark J, To A, Clouden S, Reeder G, Muldoon JJ, Chung JY, 
            Xie WH, Allain V, Steinhart Z, Chang C, Talbot A, Kim S, Rosales A, 
            Havlik LP, Pimentel H, Asokan A, Eyquem J
  TITLE     An evolved AAV variant enables efficient genetic engineering of 
            murine T cells.
  JOURNAL   Cell. 2023 Jan 19;186(2):446-460.e19. doi: 
            10.1016/j.cell.2022.12.022. Epub 2023 Jan 12.
  PUBMED    36638795
REFERENCE   2  (bases 1 to 4694)
  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: 
            "10.1016/j.cell.2022.12.022"; journalName: "Cell"; date: 
            "2023-01-19- 19"; volume: "186"; issue: "2"; pages: "446-460.e19"
COMMENT     Sequence Label: pAAV-U6/sgClta-sfGFP-Clta
FEATURES             Location/Qualifiers
     source          1..4694
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   38..178
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   49..178
                     /label=AAV2 ITR
     promoter        226..474
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     226..246
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     397..416
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        496..571
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             980..1492
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVE"
     primer_bind     complement(1019..1040)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1627..1648
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   2005..2134
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(2196..2784)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2276..2295)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2955..3746,3747..3815))
                     /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     3578..3597
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3816..3920)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3988..4006
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4044..4066)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4166..4185
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4202..4657)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4339..4360)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4551..4570
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cacagatgcg taaggagaaa ataccgcatc aggcgcccct gcaggcagct gcgcgctcgc
       61 tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc
      121 tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc
      181 ggccgctcgg tccgctagag atctccaacc ccgaggggac ccagagaggg cctatttccc
      241 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
      301 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
      361 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
      421 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgatggc
      481 cgagttggat ccattgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
      541 tcaacttgaa aaagtggcac cgagtcggtg cttttttaag cttggcgtaa ctagatgtcg
      601 acctagcaaa tggctgttcc tgatcactcc agctcccgat gctcggcgac tcccagcttc
      661 ccctagggtg gcacttccgc tggctcttcc ttctgctccc agtcacgagt tgtccccagg
      721 gctcctcccc tttgccagct gattgggcag ctcccgtctt ctgattggct gttctttttg
      781 tggctcacca acccaaccgg atatagaagc agggtttccg cttccacctt ctccctcttc
      841 tcgctcgtcc tccggtagca gccgccagca cagtgtcggc tgctgggcgg ggtgtcggcg
      901 gatcggtcgg ttggtttcgg cttcggcgtc tctgcggttg agttgggctg tcatggccgg
      961 atcgggtggg actagtggca gcaagggcga ggagctgttc accggggtgg tgcccatcct
     1021 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgcgcggcg agggcgaggg
     1081 cgatgccacc aacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     1141 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     1201 cgaccacatg aagcgccacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     1261 gcgcaccatc agcttcaagg acgacggcac ctacaagacc cgcgccgagg tgaagttcga
     1321 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     1381 catcctgggg cacaagctgg agtacaactt caacagccac aacgtctata tcaccgccga
     1441 caagcagaag aacggcatca aggccaactt caagatccgc cacaacgtgg aggacggcag
     1501 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     1561 gcccgacaac cactacctga gcacccagtc cgtgctgagc aaagacccca acgagaagcg
     1621 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactggaa ccggtgctgg
     1681 aagtggtgag ttggatccat tcggcgctcc cgccggtgcg cccggtggcc ccgctctagg
     1741 aaacggggtg gcgggcgccg gcgaagaaga cccggccgcg gccttcttgg cgcagcaaga
     1801 gagtgagatt gcgggcatcg agaacgatga ggccttcgcc atcctggacg gcggcgcccc
     1861 cgggccgcag ccacacggcg agccgccggg aggtccgggt gagagggcag cggcgttcgg
     1921 gcgggaggac ttgtgggcac actcgggtcg gtgctcttac gcggaacttg ggcttctggc
     1981 tgaagcttta acgcgtgcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     2041 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggcggcc
     2101 tcagtgagcg agcgagcgcg cagctgcctg caggacatgt gagcaaaagg ccagcaaaag
     2161 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     2221 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     2281 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     2341 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     2401 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     2461 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2521 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2581 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     2641 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2701 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2761 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2821 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2881 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     2941 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     3001 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     3061 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     3121 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     3181 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     3241 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     3301 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     3361 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     3421 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     3481 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     3541 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     3601 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     3661 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     3721 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     3781 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     3841 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3901 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     3961 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     4021 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     4081 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     4141 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     4201 aaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     4261 ttttttaacc aatagaccga aatcggcaaa atcccttata aatcaaaaga atagcccgag
     4321 atagagttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     4381 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     4441 aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     4501 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     4561 gcgaaaggag cgggcgctaa ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     4621 acacccgccg cgcttaatgc gccgctacag ggcgcgtact atggttgctt tgacgtatgc
     4681 ggtgtgaaat accg
//
