LOCUS       sequence-353740-        6566 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of the avian TVA receptor in a cre-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6566)
  TITLE     Ben-Simon lab CVS-N2c plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6566)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-DIO-EF1a-TVA-P2A-EYFP
FEATURES             Location/Qualifiers
     source          1..6566
                     /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        231..1407
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          460..1398
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1355..1375
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1437..1470)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1521..1554)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1591..2304,2305..2307))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1636..1657)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1985..2004
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2244..2265
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2308..2364)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     regulatory      2820..2829
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2836..2869
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    2920..2953
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2978..3566
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3031..3051)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3449..3460)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3600..3711
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3829..3969
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3829..3958
                     /label=AAV2 ITR
     rep_origin      4044..4499
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4131..4150)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4341..4362
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4516..4535)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4635..4657
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4695..4713)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4781..4885
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4886..4954,4955..5746)
                     /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(5104..5123)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5917..6505
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6406..6425
                     /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 gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
      361 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
      421 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc
      481 cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg
      541 ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg
      601 ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg
      661 ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct
      721 ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt
      781 gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac
      841 ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg
      901 agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg
      961 ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg
     1021 gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg
     1081 ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc
     1141 gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc
     1201 ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac
     1261 actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa
     1321 tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt
     1381 ttttttcttc catttcaggt gtcgtgaggt accggatctc tagagtcgac tccggaataa
     1441 cttcgtatag gatactttat acgaagttat gcagaatggt agctggattg tagctgctat
     1501 tagcaatatg aaacctctta ataacttcgt atagcataca ttatacgaag ttatggcgcg
     1561 ccctagggat cccctagagt cgcggccgct ttacttgtac agctcgtcca tgccgagagt
     1621 gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc
     1681 tttgctcagg gcggactggt agctcaggta gtggttgtcg ggcagcagca cggggccgtc
     1741 gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt
     1801 gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca tgatatagac
     1861 gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa
     1921 gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc
     1981 gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga cgtagccttc
     2041 gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc gggcgaagca
     2101 ctgcaggccg tagccgaagg tggtcacgag ggtgggccag ggcacgggca gcttgccggt
     2161 ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga
     2221 cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg gcaccacccc
     2281 ggtgaacagc tcctcgccct tgctcacggg accggggttt tcttccacgt ctcctgcttg
     2341 ctttaacaga gagaagttcg tggctccgga gccctcgagt tcgaacccac cccccttgga
     2401 tgcgctttca agactgaaga tgtcagacct gctttttgct ttggacttcc cccatgcagc
     2461 gataccaccc acagctacca ggcagcacag gagcactgca gtgatcagca tccacatgcg
     2521 gccgtgattc ctggctggca gagcacgtcc aggagcaggg gcagtgggag cctctgtgcc
     2581 gttgtccgtg ggcaccgcgg ggatcgcgct ggtcccgcag ccccactcgt cccgcccgtc
     2641 gtcgcagtcg gggtgtccgt cgcacagcca gtcctgcggg taacactccc cgtgggcacc
     2701 gggcggctcc gagcagcgga actgaccggg ggggcaacgg gacaaagaac cgttaccgga
     2761 cccgttaccg gtcacgttac cgggcagcag cagcagcagc agcgcgggca gcagccgcgc
     2821 catggtggcg ctagcataac ttcgtataaa gtatcctata cgaagttatt tgccttaacc
     2881 cagaaattat cactgttatt ctttagaatg gtgcaaagaa taacttcgta taatgtatgc
     2941 tatacgaagt tatgaattcg atatcaagct tatcgataat caacctctgg attacaaaat
     3001 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     3061 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     3121 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     3181 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     3241 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     3301 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     3361 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tatgttgcca cctggattct
     3421 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     3481 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     3541 gatctccctt tgggccgcct ccccgcatcg ataccgagcg ctgctcgaga gatcttcgac
     3601 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     3661 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3721 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3781 ggaagacaat agcaggcatg agatctcacg tgcggaccga gcggccgcag gaacccctag
     3841 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     3901 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     3961 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4021 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4081 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     4141 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4201 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     4261 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4321 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     4381 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     4441 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     4501 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     4561 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4621 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4681 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     4741 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     4801 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     4861 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     4921 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     4981 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5041 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5101 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5161 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5221 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5281 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5341 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     5401 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     5461 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     5521 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     5581 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     5641 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     5701 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     5761 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     5821 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     5881 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     5941 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6001 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6061 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6121 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6181 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6241 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6301 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6361 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6421 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6481 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6541 cttttgctgg ccttttgctc acatgt
//
