LOCUS       sequence-170787-        7326 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses TVA, 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 7326)
  TITLE     Salk GT3 Core Rabies Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7326)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-hSyn-FLEX-TVA-P2A-EGFP-2A-oG
FEATURES             Location/Qualifiers
     source          1..7326
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   51..191
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   62..191
                     /label=AAV2 ITR
     polyA_signal    193..417
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    425..1013
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             531..542
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     940..960
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    1015..1048
                     /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    1099..1132
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1615..1671
                     /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             join(1672..1674,1675..1677,1678..2388)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1672..1674,1675..1677,1678..2388)
                     /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(1717..1738)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1978..1997)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2325..2346
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2398..2454
                     /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(4036..4069)
                     /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(4120..4153)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      4159..4168
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4175..4622)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     repeat_region   4650..4779
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4841..5429)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4921..4940)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5600..6391,6392..6460))
                     /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     6223..6242
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6461..6565)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6633..6651
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6689..6711)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6811..6830
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6847..7302)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6984..7005)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7196..7215
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc cctgcaggca
       61 gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt
      121 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      181 taggggttcc tgccatagag cccaccgcat ccccagcatg cctgctattg tcttcccaat
      241 cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac ctactcagac
      301 aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca ccttccaggg
      361 tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa ggcacaggat
      421 ccaggcgggg aggcggccca aagggagatc cgactcgtct gagggcgaag gcgaagacgc
      481 ggaagaggcc gcagagccgg cagcaggccg cgggaaggaa ggtccgctgg attgagggcc
      541 gaagggacgt agcagaagga cgtcccgcgc agaatccagg tggcaacaca ggcgagcagc
      601 catggaaagg acgtcagctt ccccgacaac accacggaat tgtcagtgcc caacagccga
      661 gcccctgtcc agcagcgggc aaggcaggcg gcgatgagtt ccgccgtggc aatagggagg
      721 gggaaagcga aagtcccgga aaggagctga caggtggtgg caatgcccca accagtgggg
      781 gttgcgtcag caaacacagt gcacaccacg ccacgttgcc tgacaacggg ccacaactcc
      841 tcataaagag acagcaacca ggatttatac aaggaggaga aaatgaaagc catacgggaa
      901 gcaatagcat gatacaaagg cattaaagca gcgtatccac atagcgtaaa aggagcaaca
      961 tagttaagaa taccagtcaa tctttcacaa attttgtaat ccagaggttg attcataact
     1021 tcgtataaag tatcctatac gaagttattc tttgcaccat tctaaagaat aacagtgata
     1081 atttctgggt taaggcaaat aacttcgtat aatgtatgct atacgaagtt attaggtgcg
     1141 cggctgctgc ccgcgctgct gctgctgctg ctgcccggta acgtgaccgg taacgggtcc
     1201 ggtaacggtt ctttgtcccg ttgccccccc ggtcagttcc gctgctcgga gccgcccggt
     1261 gcccacgggg agtgttaccc gcaggactgg ctgtgcgacg gacaccccga ctgcgacgac
     1321 gggcgggacg agtggggctg cgggaccagc gcgatccccg cggtgcccac ggacaacggc
     1381 acagaggctc ccactgcccc tgctcctgga cgtgctctgc cagccaggaa tcacggccgc
     1441 atgtggatgc tgatcactgc agtgctcctg tgctgcctgg tagctgtggg tggtatcgct
     1501 gcatggggga agtccaaagc aaaaagcagg tctgacatct tcagtcttga aagcgcatcc
     1561 aaggagctgc tggtgcctga caagagccag gcagacttgt tctccggatc cggagctact
     1621 aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc catggtgagc
     1681 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1741 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1801 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1861 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1921 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1981 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     2041 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     2101 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     2161 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     2221 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2281 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     2341 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg atccggagct
     2401 actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg acccatggtc
     2461 ccacaggctc tcctcttcgt ccctctgctc gtcttcccac tctgctttgg taaattccct
     2521 atctacacta tccccgacaa gctgggcccc tggagtccta tcgatattca ccatctgtca
     2581 tgccctaaca atctcgtggt cgaggacgaa ggctgtacca acctgagcgg attctcctac
     2641 atggagctga aagtgggcta tatctctgct attaagatga acgggttcac atgcactggt
     2701 gtggtcaccg aggcagaaac ctacacaaat tttgtgggat atgtcaccac aactttcaag
     2761 agaaaacact ttcggccaac accagacgcc tgtcgggcag cttacaactg gaagatggct
     2821 ggcgaccccc gctatgagga aagcctgcac aatccttacc cagactatca ttggctgcgc
     2881 actgtgaaga ccacaaaaga gagcctggtc atcatttccc catctgtcgc cgacctggac
     2941 ccctacgacc gatctctcca cagtccagtg tttcccggcg gaaactgctc cggagtggcc
     3001 gtcagctcca cttactgttc caccaaccat gattatacaa tctggatgcc tgagaatcca
     3061 aggctgggga tgtcctgcga catcttcacc aacagcaggg gcaagagggc ctcaaaaggc
     3121 agcgagactt gtggatttgt ggatgaaaga gggctgtata agagcctcaa aggtgcctgc
     3181 aagctgaaac tctgtggcgt gctgggactc cggctgatgg acggaacctg ggtcgctatg
     3241 cagacatcta acgagactaa gtggtgcccc cctgggcagc tcgtgaatct gcacgacttc
     3301 cgaagcgatg agatcgaaca tctggtggtc gaggaactcg tgaagaaaag ggaggaatgt
     3361 ctcgatgctc tggagtcaat tatgactacc aagagcgtga gcttcaggag gctgagccac
     3421 ctgaggaagc tcgtccctgg cttcggaaaa gcatacacca tctttaacaa gacactgatg
     3481 gaggcagacg cccattataa atctgtgaga acctggaatg aaatcattcc tagtaaggga
     3541 tgcctgcgag tcgggggtcg atgtcaccca catgtgaacg gggtcttctt taatggtatc
     3601 attctggggc cagatggtaa cgtgctgatc cccgagatgc agtctagtct gctccagcag
     3661 cacatggaac tgctcgtgtc aagcgtcatt cccctgatgc atcctctcgc agacccaagc
     3721 acagtgttca aaaacggcga cgaggccgaa gattttgtgg aggtccacct gcccgacgtg
     3781 cacgaacgga tcagtggcgt cgatctcgga ctgcctaatt ggggcaagta cgtgctgctc
     3841 tccgctggag cactgactgc tctcatgctg atcattttcc tgatgacctg ctggcgacga
     3901 gtgaaccgct ccgagccaac tcagcacaat ctgaggggga ccggtagaga agtgtctgtc
     3961 acaccccaga gcggtaaaat catcagcagt tgggaaagtc ataaatctgg gggcgagaca
     4021 cggctctaag gtaccataac ttcgtatagg atactttata cgaagttatt aagaggtttc
     4081 atattgctaa tagcagctac aatccagcta ccattctgca taacttcgta tagcatacat
     4141 tatacgaagt tatattaacc atggtggcgg ctgactgcgc tctcaggcac gacacgactc
     4201 ctccgctgcc caccgcagac tgaggcagcg ctgagtcgcc ggcgccgcag cgcagatggt
     4261 cgcgcccgtg cccccctatc tcgcgcctcg cgtggtgcgg tccggctggg ccggcggcgg
     4321 cgcggacgcg accaaggtgg ccgggaaggg gagtttgcgg gggaccggcg agtgacgtca
     4381 gcgcgccttc agtgctgagg cggcggtggc gcgcgccgcc aggcgggggc gaaggcactg
     4441 tccgcggtgc tgaagctggc agtgcgcacg cgcctcgccg catcctgttt cccctccccc
     4501 tctctgatag gggatgcgca atttggggaa tgggggttgg gtgcttgtcc agtgggtcgg
     4561 ggtcggtcgt caggtaggca cccccacccc gcctcatcct ggtcctaaaa cccacttgca
     4621 ctcatacgca gggccctctg cagtctagca ggaaccccta gtgatggagt tggccactcc
     4681 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     4741 cggcctcagt gagcgagcga gcgcgcagct gcctgcagga catgtgagca aaaggccagc
     4801 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4861 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4921 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4981 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5041 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5101 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5161 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5221 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5281 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5341 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5401 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5461 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5521 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5581 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5641 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5701 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5761 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5821 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5881 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5941 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6001 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6061 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6121 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6181 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6241 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6301 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6361 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6421 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6481 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6541 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     6601 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     6661 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     6721 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     6781 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     6841 accataaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     6901 gctcattttt taaccaatag accgaaatcg gcaaaatccc ttataaatca aaagaatagc
     6961 ccgagataga gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     7021 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     7081 cacccaaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     7141 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     7201 agaaagcgaa aggagcgggc gctaaggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     7261 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtactatggt tgctttgacg
     7321 tatgcg
//
