LOCUS       sequence-338834-        7760 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Targeting envA-pseodotyped RVdG-CVS-N2c vectors for specific 
            retrograde labeling from cre-expressing excitatory cortical neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7760)
  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 7760)
  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: "Elife."; 
            date: "2022-08-30"; volume: "11:e79848. doi"; pages: " 
            10.7554/eLife.79848"
COMMENT     Sequence Label: pAAV-DIO-CaMKII-TVA-P2A-N2cG
FEATURES             Location/Qualifiers
     source          1..7760
                     /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        149..1437
                     /label=CaMKII promoter
                     /note="promoter for the mouse calcium/calmodulin-dependent 
                     kinase II alpha subunit"
     protein_bind    complement(1476..1509)
                     /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(1560..1593)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(3193..3249)
                     /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(3706..3729)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      3729..3738
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3745..3778
                     /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    3829..3862
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3887..4475
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3940..3960)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4358..4369)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4507..4983
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4706..4725)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5023..5163
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5023..5152
                     /label=AAV2 ITR
     rep_origin      5238..5693
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5325..5344)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5535..5556
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5710..5729)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5829..5851
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5889..5907)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5975..6079
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6080..6148,6149..6940)
                     /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(6298..6317)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7111..7699
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7600..7619
                     /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 gcgtttaaca ttatggcctt aggtcacttc atctccatgg
      181 ggttcttctt ctgattttct agaaaatgag atgggggtgc agagagcttc ctcagtgacc
      241 tgcccagggt cacatcagaa atgtcagagc tagaacttga actcagatta ctaatcttaa
      301 attccatgcc ttgggggcat gcaagtacga tatacagaag gagtgaactc attagggcag
      361 atgaccaatg agtttaggaa agaagagtcc agggcagggt acatctacac cacccgccca
      421 gccctgggtg agtccagcca cgttcacctc attatagttg cctctctcca gtcctacctt
      481 gacgggaagc acaagcagaa actgggacag gagccccagg agaccaaatc ttcatggtcc
      541 ctctgggagg atgggtgggg agagctgtgg cagaggcctc aggaggggcc ctgctgctca
      601 gtggtgacag ataggggtga gaaagcagac agagtcattc cgtcagcatt ctgggtctgt
      661 ttggtacttc ttctcacgct aaggtggcgg tgtgatatgc acaatggcta aaaagcaggg
      721 agagctggaa agaaacaagg acagagacag aggccaagtc aaccagacca attcccagag
      781 gaagcaaaga aaccattaca gagactacaa gggggaaggg aaggagagat gaattagctt
      841 cccctgtaaa ccttagaacc cagctgttgc cagggcaacg gggcaatacc tgtctcttca
      901 gaggagatga agttgccagg gtaactacat cctgtctttc tcaaggacca tcccagaatg
      961 tggcacccac tagccgttac catagcaact gcctctttgc cccacttaat cccatcccgt
     1021 ctgttaaaag ggccctatag ttggaggtgg gggaggtagg aagagcgatg atcacttgtg
     1081 gactaagttt gttcgcatcc ccttctccaa ccccctcagt acatcaccct gggggaacag
     1141 ggtccacttg ctcctgggcc cacacagtcc tgcagtattg tgtatataag gccagggcaa
     1201 agaggagcag gttttaaagt gaaaggcagg caggtgttgg ggaggcagtt accggggcaa
     1261 cgggaacagg gcgtttcgga ggtggttgcc atggggacct ggatgctgac gaaggctcgc
     1321 gaggctgtga gcagccacag tgccctgctc agaagcccca agctcgtcag tcaagccggt
     1381 tctccgtttg cactcaggag cacgggcagg cgagtggccc ctagttctgg gggcagctct
     1441 agagcggtac cggatcctct agagtcgact ccggaataac ttcgtatagg atactttata
     1501 cgaagttatg cagaatggta gctggattgt agctgctatt agcaatatga aacctcttaa
     1561 taacttcgta tagcatacat tatacgaagt tatggcgcgc cttacttgta catctcacag
     1621 tctgatctca cctccactct tatatgattc ccatgaaggt atgacttttc cgctttggga
     1681 agtgactgac acattccccc ctgtccctcc aaaactgcgt tgtttcgatt ctggtcgatt
     1741 ggctcttctg caccatgtca ttagggaaaa tatcaacacc aggccaatca tggcccctgc
     1801 agtcatcaat acatactttc cccagttcgg gagacccagg tcaacccctg agatctgttt
     1861 gtacacatcg gggaggtgaa cttcaacaaa atcctcagcc tcatcacctt ctttgaaaac
     1921 tgtagaaggg tctgccaggg ggtgcatcag ggggataact gaagattcca acaactccat
     1981 atgttgctgg aggagggatg attgcatctc tgggattagg acatggtcgt caggccctaa
     2041 tattatacca ttgaaaaaca ccccgttcac atgaggatgg caccttcctc caactttcaa
     2101 acaccctttt gaggggatga tctcattcca ggtccggact gacttgtagt gagcatcagc
     2161 ctccatcaag gttttgttga atatggtata tgcttttcca aaccctggga caagttttct
     2221 caggtgactg agacgtctga aacttactga cttggtggtc atgatggact ctaatgcgtc
     2281 cagacattcc tctcttttct tgactaactc ctccacaacg agatgctcaa tctcgtctga
     2341 gcgaaagtcg tgcaaattca ccaactgatc tggagggcac catttggtct catctgatgt
     2401 ttgcatcgcg acccatgttc catccataag tctaagtcca agaactccac ataacttgag
     2461 cctgcatgct ccttttagag acttatacag gcctctttca tccacaaagc cgcaagtctt
     2521 gttcccgttg gatgctctct tccctctgct attggtaaaa atgtcacaag gtgtccttgg
     2581 tctcggattc tcgggcatcc aaatggtgta atcatggtta gttgagcagt aggtagagga
     2641 caccgttatt cctgagcact ttccgccagg gaagaccctt gagtgaaggg atttgtcata
     2701 tgggtccaaa tctgtcacac ttggggatat gataatgagg gactctttgg tggttcttac
     2761 agttcgaagc cagtggtagt cggggtatgg attgtgtagg gactcttcat atctggggtc
     2821 accggccatc ttccagttat acgcggctct acatgcgtct ggggtggggc ggaaatgctt
     2881 tctcttgaat gtggttgtga cataaccaac aaagttggtg taggtctctg cctctgtcac
     2941 aacacctgtg caagtgaacc cgttcacttt gatggctgag atgtatccca ctttgagttc
     3001 catgtaggag aactcggaca ggttggtaca tccttcatcc tccacaacca ggttatttgg
     3061 acagctgaga tggtgtatgt caatagggct ccagggacca agttcgtctg gtatcgtgta
     3121 aatggggaac ttcccgaaac acaacgaaaa acccagaagg agtacaaaca aaagaacctg
     3181 aggaaccatg gttggcccgg gattctcttc gacatcccct gcttgtttca acagggagaa
     3241 gttagtggct ccgcttccgt tgtcttcgaa cccacccccc ttggatgcgc tttcaagact
     3301 gaagatgtca gacctgcttt ttgctttgga cttcccccat gcagcgatac cacccacagc
     3361 taccaggcag cacaggagca ctgcagtgat cagcatccac atgcggccgt gattcctggc
     3421 tggcagagca cgtccaggag caggggcagt gggagcctct gtgccgttgt ccgtgggcac
     3481 cgcggggatc gcgctggtcc cgcagcccca ctcgtcccgc ccgtcgtcgc agtcggggtg
     3541 tccgtcgcac agccagtcct gcgggtaaca ctccccgtgg gcaccgggcg gctccgagca
     3601 gcggaactga ccgggggggc aacgggacaa agaaccgtta ccggacccgt taccggtcac
     3661 gttaccgggc agcagcagca gcagcagcgc gggcagcagc cgcgccttgt cgtcatcgtc
     3721 tttgtagtcc atggtggcac tagcataact tcgtataaag tatcctatac gaagttattt
     3781 gccttaaccc agaaattatc actgttattc tttagaatgg tgcaaagaat aacttcgtat
     3841 aatgtatgct atacgaagtt atgaattcga tatcaagctt atcgataatc aacctctgga
     3901 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     3961 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     4021 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     4081 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     4141 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     4201 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     4261 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct atgttgccac
     4321 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     4381 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     4441 gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc tgctcgagag
     4501 atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc
     4561 actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag
     4621 gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg
     4681 aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa
     4741 tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc
     4801 gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag
     4861 agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac
     4921 ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc
     4981 cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg
     5041 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     5101 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     5161 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     5221 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5281 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     5341 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5401 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     5461 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5521 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     5581 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     5641 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     5701 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     5761 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     5821 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     5881 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     5941 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     6001 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     6061 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     6121 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     6181 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     6241 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     6301 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     6361 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     6421 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     6481 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     6541 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     6601 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     6661 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     6721 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     6781 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     6841 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     6901 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     6961 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     7021 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     7081 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     7141 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     7201 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     7261 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     7321 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     7381 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     7441 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     7501 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     7561 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     7621 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     7681 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     7741 ctggcctttt gctcacatgt
//
