LOCUS       sequence-228110-        8412 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Permits retrograde transsynaptic spread by driving Cre-dependent 
            co-expression of the RbG plus a nuclear reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8412)
  AUTHORS   Faget L, Osakada F, Duan J, Ressler R, Johnson AB, Proudfoot JA, Yoo
            JH, Callaway EM, Hnasko TS
  TITLE     Afferent Inputs to Neurotransmitter-Defined Cell Types in the 
            Ventral Tegmental Area.
  JOURNAL   Cell Rep. 2016 Jun 21;15(12):2796-808. doi: 
            10.1016/j.celrep.2016.05.057. Epub 2016 Jun 9.
  PUBMED    27292633
REFERENCE   2  (bases 1 to 8412)
  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"; doi: 
            "10.1016/j.celrep.2016.05.057"; journalName: "Cell Rep"; date: 
            "2016-06-21- 21"; volume: "15"; issue: "12"; pages: "2796-808"
COMMENT     Sequence Label: pAAV-Ef1a-DIO-hBFP-2A-RVG-WPRE
FEATURES             Location/Qualifiers
     source          1..8412
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1450..1483)
                     /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(1534..1567)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(3163..3216)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(3226..3291)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(3292..3990)
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             complement(4009..4386)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     protein_bind    4397..4430
                     /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    4481..4514
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4539..5127
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4592..4612)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5010..5021)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5159..5635
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5358..5377)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5675..5815
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5675..5804
                     /label=AAV2 ITR
     rep_origin      5890..6345
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5977..5996)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6187..6208
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6362..6381)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6481..6503
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6541..6559)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6627..6731
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6732..6800,6801..7592)
                     /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(6950..6969)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7763..8351
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8252..8271
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc
     1441 gactccggaa taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga
     1501 ttgtagctgc tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg
     1561 aagttatggc gcgccttaca gtcgggtttc gccgcctgat ttatggcttt cccaactact
     1621 aatgatcttg cctgactgag gtgtgactga cacttctctc ccggttcctc gcagattgtg
     1681 ctgagtgggc tcactccggt tcacgcgccg acagcaggtc atcaggaaaa tgatcagcat
     1741 cagggcagtc agtgctccag cactcagcag cacgtacttg ccccaatttg gcagtcccag
     1801 gtccacgcca gacacctggt tatgcacatc aggcaggtgg acctccacaa agtcttcggc
     1861 ctcatctccg tctttgaaca ctgtggaggg atctgccagt ggatggacca gaggaatcac
     1921 gcttgattcc agcagctcca tgtgctgctg cagcagacta gactgcatct cagggatcag
     1981 cacgttgccg tcgggcccca gaatgattcc attaaagaag acgccgttca catgtgggtg
     2041 acagcgtccg cccactcgca ggcagccctt gcttggcaga atttcattcc aggtgcgcac
     2101 tgatttataa tgggcgtctg cctccatcag tgtcttgtta aagatggtgt aggctttgcc
     2161 gaagccgggc accagctttc gcaggtgaga cagcctcctg aagctcacgc tcttggtagt
     2221 catgatggat tccagggcat ccagacattc ctcccttttt cgcaccagtt cctcgaccac
     2281 cagatgttcg atctcatcgg atctgaagtc gtgcagattc accagtttgt caggggggca
     2341 ccacttagtc tcgtttgatg tctgcatact gacccaggtg ccatccatca gtctcagtcc
     2401 cagcacgcca cacagtttca gcttgcaagc ccctttcagg ctcttataca gtcccctttc
     2461 gtccacaaac ccacaagtct cgcttccttt tgaggctcgc ttgccgcggg aatttgtgaa
     2521 aatgtcgcag ctcatcccca gcctgggatt ctctggcatc cagattgtat aatcatggtt
     2581 ggtgctacag taagtggagc tgacagccac cccagagcac tttccggagg gaaacacccg
     2641 actgtgcaga gatctatcgt atggatccag gtctgcgaca gaaggggaaa tgatgaccag
     2701 ggactctttt gtggtcttca cagtccgcag ccatctatag tctgggtaag gattgtgcag
     2761 agattcctca tatcgtggat cgccagccat cttccagttg taagcggcgc gacaggcgtc
     2821 gggtgttggt ctaaagtgtt tcctcttgaa agttgtggtg acatagccca caaaatttgt
     2881 gtaggtttct gcctcggtga ccacgccagt gcatgtgaac ccgttgacct taatagccag
     2941 gatatatccc actttcagct ccatgtagga gaagccgctc aggttggtac acccttcgtc
     3001 ctcgaccacc agattgttag ggcaactcag atggtgaata tcgataggag accaggggcc
     3061 cagtttatca ggaatagtgt agattgggaa cttgccgaag cacagtggaa agaccagcag
     3121 agggacaaac agcagagcct gggggaccat gctgccgggg gcggggccgg ggttctcctc
     3181 cacgtcgccg caggtcagca ggctgccgcg gccctcacca ctacccttgt catcgtcatc
     3241 cttgtagtcg atgtcatgat ctttataatc accgtcatgg tctttgtagt cattaagctt
     3301 gtgccccagt ttgctaggga ggtcgcagta tctggccact gccacctcgt gctgctcgac
     3361 gtaggtctcg ttgttggcct ccttgattct ttccagtctg tagtccacat agtagacgcc
     3421 aggcatcttg aggttcttag cgggtttctt ggatctatat gtggtcttga tgtttgcgat
     3481 cagatggctc ccgcccacga gcttcagggc catgtcgttt ctgccttcca ggccgccgtc
     3541 agcggggtac agcgtctcgg tgaaggcctc ccagccgagt gttttcttct gcatcacagg
     3601 gccgttggat gtgaagttca cccctctgat cttgacgttg tagatgaggc agccgtcctg
     3661 gaggctggtg tcctgggtag cggtcagcac gcccccgtct tcgtatgtgg tgactctctc
     3721 ccatgtgaag ccctcaggga aggactgctt gaagaagtcg gggatgccct gggtgtggtt
     3781 gatgaaggtc ttgctgccgt agaggaagct agtagccagg atgtcgaagg cgaaggggag
     3841 agggccgccc tcgaccacct tgattctcat ggtctgggtg ccctcgtagg gcttgccttc
     3901 gccctcggat gtgcacttga agtgatggtt gtccacggtg ccctccatgt acagcttcat
     3961 gtgcatgttc tccttaatca gctcgctcat ggtggcgacc ggtggatcct tagcgctggt
     4021 gtacttggtg atggccttag taccctcgga cacggcgtgc ttggccaact ccccaggcag
     4081 cagcaggcgc acggccgtct ggatctccct ggaggtgatg gtcgagcgct tgttgtaatg
     4141 cgccaggcgg gaagcctcac ctgcgatgcg ctcgaaaatg tcgttcacaa acgaattcat
     4201 gatgcccatg gccttggacg aaatgccggt gtcagggtgg acctgcttca gaaccttgta
     4261 cacatagatg gaatagctct ccttgcggct gcgcttgcgc ttcttgccgc ctttcttctg
     4321 cgccttagtc accgccttct tggagccctt tttcggggcg ggagcagact tcgctggctc
     4381 tggcatggtg gctagcataa cttcgtataa agtatcctat acgaagttat ttgccttaac
     4441 ccagaaatta tcactgttat tctttagaat ggtgcaaaga ataacttcgt ataatgtatg
     4501 ctatacgaag ttatgaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa
     4561 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     4621 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     4681 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     4741 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     4801 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     4861 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     4921 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc
     4981 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     5041 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     5101 ggatctccct ttgggccgcc tccccgcatc gataccgagc gctgctcgag agatctacgg
     5161 gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt
     5221 gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt
     5281 ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac
     5341 ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct
     5401 cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt
     5461 gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg
     5521 tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg
     5581 gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat
     5641 tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc
     5701 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     5761 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     5821 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     5881 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     5941 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     6001 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     6061 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     6121 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     6181 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt
     6241 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     6301 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     6361 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     6421 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     6481 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     6541 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     6601 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     6661 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     6721 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     6781 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     6841 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     6901 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     6961 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     7021 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     7081 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     7141 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     7201 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     7261 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     7321 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     7381 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     7441 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     7501 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     7561 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     7621 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     7681 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     7741 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     7801 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     7861 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     7921 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     7981 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     8041 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     8101 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     8161 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     8221 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     8281 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     8341 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     8401 ttgctcacat gt
//
