LOCUS       sequence-350222-        7787 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting envA-pseodotyped RVdG-CVS-N2c vectors for specific 
            retrograde labeling from Flp-expressing neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7787)
  AUTHORS   Sumser A, Joesch M, Jonas P, Ben-Simon Y
  TITLE     Fast, high-throughput production of improved rabies viral vectors 
            for specific, efficient and versatile transsynaptic retrograde 
            labeling.
  JOURNAL   Elife. 2022 Aug 30;11:e79848. doi: 10.7554/eLife.79848.
  PUBMED    36040301
REFERENCE   2  (bases 1 to 7787)
  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: "Elife."; 
            date: "2022-08-30"; volume: "11:e79848. doi"; pages: " 
            10.7554/eLife.79848"
COMMENT     Sequence Label: pAAV-FRT-EF1a-TVA-2A-N2cG
FEATURES             Location/Qualifiers
     source          1..7787
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   110..250
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   110..239
                     /label=AAV2 ITR
     rep_origin      325..780
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(412..431)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     622..643
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(797..816)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     916..938
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(976..994)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1062..1166
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1167..1235,1236..2027)
                     /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(1385..1404)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2198..2786
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2687..2706
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2848..2977
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        3078..4256
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3309..4247
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4204..4224
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    4287..4320
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(6004..6060)
                     /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(6082..6147)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      6603..6612
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(6619..6652)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    6761..7349
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6814..6834)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7232..7243)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    join(7381..7787,1..70)
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(7580..7599)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
ORIGIN
        1 gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt
       61 ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg agcggccgca ggaaccccta
      121 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
      181 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
      241 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
      301 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
      361 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
      421 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
      481 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
      541 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
      601 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
      661 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
      721 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
      781 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
      841 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
      901 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
      961 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     1021 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1081 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1141 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     1201 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     1261 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     1321 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     1381 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     1441 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     1501 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     1561 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     1621 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     1681 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     1741 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     1801 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     1861 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     1921 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     1981 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2041 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     2101 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2161 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2221 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2281 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2341 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2401 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2461 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2521 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2581 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2641 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2701 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2761 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2821 ccttttgctg gccttttgct cacatgtcct gcaggcagct gcgcgctcgc tcgctcactg
     2881 aggccgcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc
     2941 agagagggag tggccaactc catcactagg ggttcctgcg gccgcacgcg taagctttgc
     3001 aaagatggat aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct
     3061 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
     3121 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg
     3181 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
     3241 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
     3301 gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc
     3361 cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc
     3421 gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg
     3481 cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc
     3541 gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg
     3601 cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta
     3661 tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg
     3721 cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc
     3781 ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc
     3841 tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag
     3901 ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa
     3961 ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc
     4021 cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg
     4081 aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag
     4141 cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca
     4201 ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgaggta
     4261 ccggatcctc tagagtcgac tccggagaag ttcctattct ctagaaagta taggaacttc
     4321 gcagaatggt agctggattg tagctgctat tagcaatatg aaacctctta gaagttccta
     4381 ttcttcaaaa ggtataggaa cttcggcgcg ccttacttgt acatcttaca gtctgatctc
     4441 acctccactc ttatatgatt cccatgaagg tatgactttt ccgctttggg aagtgactga
     4501 cacattcccc cctgtccctc caaaactgcg ttgtttcgat tctggtcgat tggctcttct
     4561 gcaccatgtc attagggaaa atatcaacac caggccaatc atggcccctg cagtcatcaa
     4621 tacatacttt ccccagttcg ggagacccag gtcaacccct gagatctgtt tgtacacatc
     4681 ggggaggtga acttcaacaa aatcctcagc ctcatcacct tctttgaaaa ctgtagaagg
     4741 gtctgccagg gggtgcatca gggggataac tgaagattcc aacaactcca tatgttgctg
     4801 gaggagggat gattgcatct ctgggattag gacatggtcg tcaggcccta atattatacc
     4861 attgaaaaac accccgttca catgaggatg gcaccttcct ccaactttca aacacccttt
     4921 tgaggggatg atctcattcc aggtccggac tgacttgtag tgagcatcag cctccatcaa
     4981 ggttttgttg aatatggtat atgcttttcc aaaccctggg acaagttttc tcaggtgact
     5041 gagacgtctg aaacttactg acttggtggt catgatggac tctaatgcgt ccagacattc
     5101 ctctcttttc ttgactaact cctccacaac gagatgctca atctcgtctg agcgaaagtc
     5161 gtgcaaattc accaactgat ctggagggca ccatttggtc tcatctgatg tttgcatcgc
     5221 gacccatgtt ccatccataa gtctaagtcc aagaactcca cataacttga gcctgcatgc
     5281 tccttttaga gacttataca ggcctctttc atccacaaag ccgcaagtct tgttcccgtt
     5341 ggatgctctc ttccctctgc tattggtaaa aatgtcacaa ggtgtccttg gtctcggatt
     5401 ctcgggcatc caaatggtgt aatcatggtt agttgagcag taggtagagg acaccgttat
     5461 tcctgagcac tttccgccag ggaagaccct tgagtgaagg gatttgtcat atgggtccaa
     5521 atctgtcaca cttggggata tgataatgag ggactctttg gtggttctta cagttcgaag
     5581 ccagtggtag tcggggtatg gattgtgtag ggactcttca tatctggggt caccggccat
     5641 cttccagtta tacgcggctc tacatgcgtc tggggtgggg cggaaatgct ttctcttgaa
     5701 tgtggttgtg acataaccaa caaagttggt gtaggtctct gcctctgtca caacacctgt
     5761 gcaagtgaac ccgttcactt tgatggctga gatgtatccc actttgagtt ccatgtagga
     5821 gaactcggac aggttggtac atccttcatc ctccacaacc aggttatttg gacagctgag
     5881 atggtgtatg tcaatagggc tccagggacc aagttcgtct ggtatcgtgt aaatggggaa
     5941 cttcccgaaa cacaacgaaa aacccagaag gagtacaaac aaaagaacct gaggaaccat
     6001 ggttggcccg ggattctctt cgacatcccc tgcttgtttc aacagggaga agttagtggc
     6061 tccgcttccg ttgtcttcga acttgtcgtc atcgtctttg tagtcgatat catgatcctt
     6121 ataatcacca tcatgatcct tataatcgcc ggtgctgccc ggttcgaacc cacccccctt
     6181 ggatgcgctt tcaagactga agatgtcaga cctgcttttt gctttggact tcccccatgc
     6241 agcgatacca cccacagcta ccaggcagca caggagcact gcagtgatca gcatccacat
     6301 gcggccgtga ttcctggctg gcagagcacg tccaggagca ggggcagtgg gagcctctgt
     6361 gccgttgtcc gtgggcaccg cggggatcgc gctggtcccg cagccccact cgtcccgccc
     6421 gtcgtcgcag tcggggtgtc cgtcgcacag ccagtcctgc gggtaacact ccccgtgggc
     6481 accgggcggc tccgagcagc ggaactgacc gggggggcaa cgggacaaag aaccgttacc
     6541 ggacccgtta ccggtcacgt taccgggcag cagcagcagc agcagcgcgg gcagcagccg
     6601 cgccatggtg gcactagcga agttcctata ctttctagag aataggaact tcttgcctta
     6661 acccagaaat tatcactgtt attctttaga atggtgcaaa gagaagttcc tatacctttt
     6721 gaagaatagg aacttcgaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa
     6781 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     6841 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     6901 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     6961 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     7021 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     7081 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     7141 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat
     7201 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     7261 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     7321 tcggatctcc ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatctac
     7381 gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca
     7441 gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc
     7501 ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca
     7561 acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg
     7621 ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg
     7681 ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg
     7741 ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggt
//
