LOCUS       sequence-350218-        7499 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting envA-pseodotyped RVdG vectors for specific retrograde 
            labeling specifically from cre-expressing neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7499)
  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 7499)
  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-DIO-Ef1a-TVA-2A-N2cG
FEATURES             Location/Qualifiers
     source          1..7499
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      114..569
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(201..220)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     411..432
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(586..605)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     705..727
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(765..783)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        851..955
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(956..1024,1025..1816)
                     /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(1174..1193)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1987..2575
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2476..2495
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2637..2766
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2867..4043
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3096..4034
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3991..4011
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(4073..4106)
                     /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(4157..4190)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(5790..5846)
                     /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(5868..5933)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      6389..6398
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    6405..6438
                     /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    6489..6522
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    6547..7135
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6600..6620)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7018..7029)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    7169..7280
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   join(7398..7499,1..39)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(7398..7499,1..28)
                     /label=AAV2 ITR
ORIGIN
        1 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
       61 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
      121 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      181 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
      241 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
      301 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
      361 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
      421 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
      481 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
      541 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
      601 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
      661 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
      721 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
      781 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
      841 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
      901 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
      961 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1021 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     1081 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     1141 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     1201 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     1261 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     1321 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     1381 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     1441 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     1501 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     1561 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     1621 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     1681 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     1741 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     1801 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     1861 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     1921 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     1981 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2041 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2101 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     2161 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2221 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     2281 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     2341 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     2401 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     2461 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     2521 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     2581 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtcctg
     2641 caggcagctg cgcgctcgct cgctcactga ggccgcccgg gcgtcgggcg acctttggtc
     2701 gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg
     2761 gttcctgcgg ccgcacgcgt aagctttgca aagatggata aagttttaaa cagagaggaa
     2821 tctttgcagc taatggacct tctaggtctt gaaaggagtg ggaattggct ccggtgcccg
     2881 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
     2941 ttgccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
     3001 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     3061 ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg
     3121 ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc acctggctgc
     3181 agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt
     3241 gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc
     3301 gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag
     3361 ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa
     3421 atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg
     3481 cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa
     3541 tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt
     3601 gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa
     3661 gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag
     3721 agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt
     3781 catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgagctttt
     3841 ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg
     3901 agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg
     3961 ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt
     4021 ttcttccatt tcaggtgtcg tgaggtaccg gatctctaga gtcgactccg gaataacttc
     4081 gtataggata ctttatacga agttatgcag aatggtagct ggattgtagc tgctattagc
     4141 aatatgaaac ctcttaataa cttcgtatag catacattat acgaagttat ggcgcgcctt
     4201 acttgtacat cttacagtct gatctcacct ccactcttat atgattccca tgaaggtatg
     4261 acttttccgc tttgggaagt gactgacaca ttcccccctg tccctccaaa actgcgttgt
     4321 ttcgattctg gtcgattggc tcttctgcac catgtcatta gggaaaatat caacaccagg
     4381 ccaatcatgg cccctgcagt catcaataca tactttcccc agttcgggag acccaggtca
     4441 acccctgaga tctgtttgta cacatcgggg aggtgaactt caacaaaatc ctcagcctca
     4501 tcaccttctt tgaaaactgt agaagggtct gccagggggt gcatcagggg gataactgaa
     4561 gattccaaca actccatatg ttgctggagg agggatgatt gcatctctgg gattaggaca
     4621 tggtcgtcag gccctaatat tataccattg aaaaacaccc cgttcacatg aggatggcac
     4681 cttcctccaa ctttcaaaca cccttttgag gggatgatct cattccaggt ccggactgac
     4741 ttgtagtgag catcagcctc catcaaggtt ttgttgaata tggtatatgc ttttccaaac
     4801 cctgggacaa gttttctcag gtgactgaga cgtctgaaac ttactgactt ggtggtcatg
     4861 atggactcta atgcgtccag acattcctct cttttcttga ctaactcctc cacaacgaga
     4921 tgctcaatct cgtctgagcg aaagtcgtgc aaattcacca actgatctgg agggcaccat
     4981 ttggtctcat ctgatgtttg catcgcgacc catgttccat ccataagtct aagtccaaga
     5041 actccacata acttgagcct gcatgctcct tttagagact tatacaggcc tctttcatcc
     5101 acaaagccgc aagtcttgtt cccgttggat gctctcttcc ctctgctatt ggtaaaaatg
     5161 tcacaaggtg tccttggtct cggattctcg ggcatccaaa tggtgtaatc atggttagtt
     5221 gagcagtagg tagaggacac cgttattcct gagcactttc cgccagggaa gacccttgag
     5281 tgaagggatt tgtcatatgg gtccaaatct gtcacacttg gggatatgat aatgagggac
     5341 tctttggtgg ttcttacagt tcgaagccag tggtagtcgg ggtatggatt gtgtagggac
     5401 tcttcatatc tggggtcacc ggccatcttc cagttatacg cggctctaca tgcgtctggg
     5461 gtggggcgga aatgctttct cttgaatgtg gttgtgacat aaccaacaaa gttggtgtag
     5521 gtctctgcct ctgtcacaac acctgtgcaa gtgaacccgt tcactttgat ggctgagatg
     5581 tatcccactt tgagttccat gtaggagaac tcggacaggt tggtacatcc ttcatcctcc
     5641 acaaccaggt tatttggaca gctgagatgg tgtatgtcaa tagggctcca gggaccaagt
     5701 tcgtctggta tcgtgtaaat ggggaacttc ccgaaacaca acgaaaaacc cagaaggagt
     5761 acaaacaaaa gaacctgagg aaccatggtt ggcccgggat tctcttcgac atcccctgct
     5821 tgtttcaaca gggagaagtt agtggctccg cttccgttgt cttcgaactt gtcgtcatcg
     5881 tctttgtagt cgatatcatg atccttataa tcaccatcat gatccttata atcgccggtg
     5941 ctgcccggtt cgaacccacc ccccttggat gcgctttcaa gactgaagat gtcagacctg
     6001 ctttttgctt tggacttccc ccatgcagcg ataccaccca cagctaccag gcagcacagg
     6061 agcactgcag tgatcagcat ccacatgcgg ccgtgattcc tggctggcag agcacgtcca
     6121 ggagcagggg cagtgggagc ctctgtgccg ttgtccgtgg gcaccgcggg gatcgcgctg
     6181 gtcccgcagc cccactcgtc ccgcccgtcg tcgcagtcgg ggtgtccgtc gcacagccag
     6241 tcctgcgggt aacactcccc gtgggcaccg ggcggctccg agcagcggaa ctgaccgggg
     6301 gggcaacggg acaaagaacc gttaccggac ccgttaccgg tcacgttacc gggcagcagc
     6361 agcagcagca gcgcgggcag cagccgcgcc atggtggcac tagcataact tcgtataaag
     6421 tatcctatac gaagttattt gccttaaccc agaaattatc actgttattc tttagaatgg
     6481 tgcaaagaat aacttcgtat aatgtatgct atacgaagtt atgaattcga tatcaagctt
     6541 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     6601 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     6661 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     6721 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     6781 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     6841 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     6901 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     6961 ctgctcgcct atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     7021 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     7081 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     7141 taccgagcgc tgctcgagag atcttcgact gtgccttcta gttgccagcc atctgttgtt
     7201 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     7261 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     7321 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatga gatctcacgt
     7381 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     7441 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccggg
//
