LOCUS       sequence-307586-        6520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent expression of TVA:mCherry fusion protein for rabies 
            tracing..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6520)
  AUTHORS   Fischer KB, Collins HK, Callaway EM
  TITLE     Sources of off-target expression from recombinase-dependent AAV 
            vectors and mitigation with cross-over insensitive ATG-out vectors.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Dec 16. pii: 1915974116. doi: 
            10.1073/pnas.1915974116.
  PUBMED    31843925
REFERENCE   2  (bases 1 to 6520)
  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"; journalName: "Proc Natl 
            Acad Sci U S A. 2019 Dec 16. pii: 1915974116. doi: 
            10.1073/pnas.1915974116."
COMMENT     Sequence Label: pAAV-nef-CIAO-TVA-mCherry
FEATURES             Location/Qualifiers
     source          1..6520
                     /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        177..388
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             401..669
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      700..709
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(711..744)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     intron          1265..1330
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     polyA_signal    1908..2042
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1945..1964)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1999..2018
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(2062..2095)
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2537..3244
                     /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(2679..2697)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2947..2966
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3267..3743
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3466..3485)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3783..3923
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3783..3912
                     /label=AAV2 ITR
     rep_origin      3998..4453
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4085..4104)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4295..4316
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4470..4489)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4589..4611
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4649..4667)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4735..4839
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4840..4908,4909..5700)
                     /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(5058..5077)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5871..6459
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6360..6379
                     /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 gcgtaaggat ctgcgatcgc tccggtgccc gtcagtgggc
      181 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg
      241 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
      301 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
      361 tcgcaacggg tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc
      421 acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc
      481 tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc
      541 gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac
      601 gctttgcctg accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg
      661 ttacagatcc aagctgtgac cggcgcctac ggatccgccg ccaccatggg taccgttcgt
      721 atagcataca ttatacgaag ttatgagctc tcggggacac caaatatggc gatctcggcc
      781 ttttcgtttc ttggagctgg gacatgtttg ccatcgatcc atctaccacc agaacggccg
      841 ttagatctgc tgccaccgtt gtttccaccg aagaaaccac cgttgccgta accaccacga
      901 cggttgttgc taaagaagct gccaccgcca cggccaccgt tgtagccgcc gttgttgtta
      961 ttgtagttgc tactgttatt tctggcactt cttggttttc ctcttaagtg aggaggaaca
     1021 taaccattct cgttgttgtc gttgatgctt aaattttgca cttgttcgct cagttcagcc
     1081 ataatatgaa atgcttttct tttgttctta cggaatacca cttgccacct atcaccacaa
     1141 ctaacttttt cccgttcctc catctctttt atattttttt tctcgaggga tctttgtgaa
     1201 ggaaccttac ttctgtggtg tgacataatt ggacaaacta cctacagaga tttaaagctc
     1261 taaggtaaat ataaaatttt taagtgtata atgtgttaaa ctactgattc taattgtttg
     1321 tgtattttag attccaacct atggaactga tgaatgggag cagtggtgga atgcctttaa
     1381 tgaggaaaac ctgttttgct cagaagaaat gccatctagt gatgatgagg ctactgctga
     1441 ctctcaacat tctactcctc caaaaaagaa gaagagaaag gtagaagacc ccaaggactt
     1501 tccttcagaa ttgctaagtt ttttgagtca tgctgtgttt agtaatagaa ctcttgcttg
     1561 ctttgctatt tacaccacaa aggaaaaagc tgcactgcta tacaagaaaa ttatggaaaa
     1621 atattctgta acctttataa gtaggcataa cagttataat cataacatac tgttttttct
     1681 tactccacac aggcatagag tgtctgctat taataactat gctcaaaaat tgtgtacctt
     1741 tagcttttta atttgtaaag gggttaataa ggaatatttg atgtatagtg ccttgactag
     1801 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     1861 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     1921 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     1981 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     2041 tctgacatgg taagtgaatt cataacttcg tatagcatac attatacgaa cggtagctag
     2101 cgcgcggctg ctgcccgcgc tgctgctgct gctgctgccc ggtaacgtga ccggtaacgg
     2161 gtccggtaac ggttctttgt cccgttgccc ccccggtcag ttccgctgct cggagccgcc
     2221 cggtgcccac ggggagtgtt acccgcagga ctggctgtgc gacggacacc ccgactgcga
     2281 cgacgggcgg gacgagtggg gctgcgggac cagcgcgatc cccgcggtgc ccacggacaa
     2341 cggcacagag gctcccactg cccctgctcc tggacgtgct ctgccagcca ggaatcacgg
     2401 ccgcatgtgg atgctgatca ctgcagtgct cctgtgctgc ctggtagctg tgggtggtat
     2461 cgctgcatgg gggaagtcca aagcaaaaag caggtctgac atcttcagtc ttgaaagcgc
     2521 atccaagggg ggtggggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     2581 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     2641 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     2701 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     2761 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     2821 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     2881 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     2941 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     3001 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     3061 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     3121 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     3181 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     3241 gtaaggcgcg ccctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc
     3301 tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt
     3361 gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat
     3421 ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa
     3481 gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga
     3541 ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct
     3601 aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact
     3661 cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa
     3721 ccactgctcc cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc
     3781 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     3841 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     3901 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     3961 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     4021 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     4081 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4141 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4201 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     4261 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4321 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     4381 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     4441 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4501 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4561 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     4621 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     4681 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     4741 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     4801 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     4861 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     4921 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     4981 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5041 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5101 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5161 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5221 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5281 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5341 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5401 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     5461 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5521 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     5581 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     5641 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     5701 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     5761 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     5821 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     5881 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     5941 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6001 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6061 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6121 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6181 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6241 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6301 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6361 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6421 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6481 ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
