LOCUS       sequence-354737-        7293 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TVA(TC66T), EGFP, and optimized Rabies G (oG) protein in a
            FLEX cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7293)
  TITLE     AAV plasmids to express TVA and optimized Rabies G protein
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7293)
  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            "Unpublished"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: AAV-DIO-TC66T-2A-EGFP-2A-oG
FEATURES             Location/Qualifiers
     source          1..7293
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        104..551
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      558..567
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    573..606
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    657..690
                     /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)."
     CDS             complement(2272..2328)
                     /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"
     CDS             complement(join(2338..3048,3049..3051,3052..3054))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2338..3048,3049..3051,3052..3054))
                     /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(2380..2401)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2729..2748
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2988..3009
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(3055..3111)
                     /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"
     protein_bind    complement(3561..3594)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(3645..3678)
                     /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)."
     misc_feature    3680..4268
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3733..3753)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4151..4162)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4276..4500
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4502..4642
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4502..4631
                     /label=AAV2 ITR
     rep_origin      4717..5172
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4804..4823)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5014..5035
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5189..5208)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5308..5330
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5368..5386)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5454..5558
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5559..5627,5628..6419)
                     /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(5777..5796)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6590..7178
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7079..7098
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(7240..7293,1..76)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
       61 atcactaggg gttcctgcta gactgcagag ggccctgcgt atgagtgcaa gtgggtttta
      121 ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac ccactggaca
      181 agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg aggggaaaca
      241 ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg ccttcgcccc
      301 cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg tcactcgccg
      361 gtcccccgca aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc gccggcccag
      421 ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat ctgcgctgcg
      481 gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggaggagtc gtgtcgtgcc
      541 tgagagcgca gtcagccgcc accatggtta atataacttc gtataatgta tgctatacga
      601 agttatgcag aatggtagct ggattgtagc tgctattagc aatatgaaac ctcttaataa
      661 cttcgtataa agtatcctat acgaagttat ggtaccttag agccgtgtct cgcccccaga
      721 tttatgactt tcccaactgc tgatgatttt accgctctgg ggtgtgacag acacttctct
      781 accggtcccc ctcagattgt gctgagttgg ctcggagcgg ttcactcgtc gccagcaggt
      841 catcaggaaa atgatcagca tgagagcagt cagtgctcca gcggagagca gcacgtactt
      901 gccccaatta ggcagtccga gatcgacgcc actgatccgt tcgtgcacgt cgggcaggtg
      961 gacctccaca aaatcttcgg cctcgtcgcc gtttttgaac actgtgcttg ggtctgcgag
     1021 aggatgcatc aggggaatga cgcttgacac gagcagttcc atgtgctgct ggagcagact
     1081 agactgcatc tcggggatca gcacgttacc atctggcccc agaatgatac cattaaagaa
     1141 gaccccgttc acatgtgggt gacatcgacc cccgactcgc aggcatccct tactaggaat
     1201 gatttcattc caggttctca cagatttata atgggcgtct gcctccatca gtgtcttgtt
     1261 aaagatggtg tatgcttttc cgaagccagg gacgagcttc ctcaggtggc tcagcctcct
     1321 gaagctcacg ctcttggtag tcataattga ctccagagca tcgagacatt cctccctttt
     1381 cttcacgagt tcctcgacca ccagatgttc gatctcatcg cttcggaagt cgtgcagatt
     1441 cacgagctgc ccaggggggc accacttagt ctcgttagat gtctgcatag cgacccaggt
     1501 tccgtccatc agccggagtc ccagcacgcc acagagtttc agcttgcagg cacctttgag
     1561 gctcttatac agccctcttt catccacaaa tccacaagtc tcgctgcctt ttgaggccct
     1621 cttgcccctg ctgttggtga agatgtcgca ggacatcccc agccttggat tctcaggcat
     1681 ccagattgta taatcatggt tggtggaaca gtaagtggag ctgacggcca ctccggagca
     1741 gtttccgccg ggaaacactg gactgtggag agatcggtcg taggggtcca ggtcggcgac
     1801 agatggggaa atgatgacca ggctctcttt tgtggtcttc acagtgcgca gccaatgata
     1861 gtctgggtaa ggattgtgca ggctttcctc atagcggggg tcgccagcca tcttccagtt
     1921 gtaagctgcc cgacaggcgt ctggtgttgg ccgaaagtgt tttctcttga aagttgtggt
     1981 gacatatccc acaaaatttg tgtaggtttc tgcctcggtg accacaccag tgcatgtgaa
     2041 cccgttcatc ttaatagcag agatatagcc cactttcagc tccatgtagg agaatccgct
     2101 caggttggta cagccttcgt cctcgaccac gagattgtta gggcatgaca gatggtgaat
     2161 atcgatagga ctccaggggc ccagcttgtc ggggatagtg tagataggga atttaccaaa
     2221 gcagagtggg aagacgagca gagggacgaa gaggagagcc tgtgggacca tgggtccagg
     2281 gttctcctcc acgtctccag cctgcttcag caggctgaag ttagtagctc cggatccctt
     2341 gtacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca gcaggaccat
     2401 gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac tgggtgctca ggtagtggtt
     2461 gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag
     2521 ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga tgccgttctt
     2581 ctgcttgtcg gccatgatat agacgttgtg gctgttgtag ttgtactcca gcttgtgccc
     2641 caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt
     2701 gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat
     2761 ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat
     2821 gtggtcgggg tagcggctga agcactgcac gccgtaggtc agggtggtca cgagggtggg
     2881 ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt
     2941 ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag
     3001 ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca ccatgggtcc
     3061 agggttctcc tccacgtctc cagcctgctt cagcaggctg aagttagtag ctccggatcc
     3121 cccacccccc ttggatgcgc tttcaagact gaagatgtca gacctgcttt ttgctttgga
     3181 cttcccccat gcagcgatac cacccacagc taccaggcag cacaggagca ctgcagtgat
     3241 cagcatccac atgcggccgt gattcctggc tggcagagca cgtccaggag caggggcagt
     3301 gggagcctct gtgccgttgt ccgtgggcac cgcggggatc gcgctggtcc cgcagcccca
     3361 ggtgtcccgc ccgtcgtcgc agtcggggtg tccgtcgcac agccagtcct gcgggtaaca
     3421 ctccccgtgg gcaccgggcg gctccgagca gcggaactga ccgggggggc aacgggacaa
     3481 agaaccgtta ccggacccgt taccggtcac gttaccgggc agcagcagca gcagcagcgc
     3541 gggcagcagc cgcgcaccta ataacttcgt atagcataca ttatacgaag ttatttgcct
     3601 taacccagaa attatcactg ttattcttta gaatggtgca aagaataact tcgtatagga
     3661 tactttatac gaagttatga atcaacctct ggattacaaa atttgtgaaa gattgactgg
     3721 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     3781 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     3841 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     3901 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     3961 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     4021 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac
     4081 gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     4141 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     4201 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     4261 ctccccgcct ggatcctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     4321 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     4381 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     4441 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     4501 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     4561 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     4621 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     4681 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     4741 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     4801 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4861 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4921 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4981 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     5041 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     5101 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     5161 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     5221 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     5281 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5341 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     5401 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     5461 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5521 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5581 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5641 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5701 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5761 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5821 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5881 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5941 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6001 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6061 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6121 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6181 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6241 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6301 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6361 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6421 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6481 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6541 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6601 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6661 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6721 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6781 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6841 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6901 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6961 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     7021 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7081 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7141 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7201 tacggttcct ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc
     7261 gctcgctcac tgaggccgcc cgggcgtcgg gcg
//
