LOCUS Exported 8402 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Permits retrograde transsynaptic spread by driving Cre-dependent co-expression of the RbG plus a nuclear reporter. ACCESSION . VERSION . KEYWORDS pAAV-Ef1a-DIO-hBFP-2A-RVG-WPRE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8402) 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 8402) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8402 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 120..249 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" promoter 350..1528 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 581..1519 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1476..1496 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" protein_bind 1559..1592 /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind 1643..1676 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(3272..3325) /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(3335..3400) /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS complement(3401..4099) /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC DLPSKLGHKLN" CDS complement(4118..4495) /codon_start=1 /gene="HIST1H2BJ" /product="human histone H2B" /label=H2B /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP GELAKHAVSEGTKAITKYTSAK" protein_bind complement(4506..4539) /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind complement(4590..4623) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 4648..5236 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(4701..4721) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5119..5130) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 5268..5744 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(5467..5486) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 5784..5924 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5784..5913 /label=AAV2 ITR rep_origin 5999..6454 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6086..6105) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6296..6317 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6471..6490) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6590..6612 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6650..6668) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6736..6840 /gene="bla" /label=AmpR promoter CDS 6841..7701 /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(7059..7078) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7872..58 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8361..8380 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 61 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtc 121 ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg 181 gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta 241 ggggttcctg cggccgcacg cgtaagcttt gcaaagatgg ataaagtttt aaacagagag 301 gaatctttgc agctaatgga ccttctaggt cttgaaagga gtgggaattg gctccggtgc 361 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg 421 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta 481 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt 541 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc 601 ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacctg 661 gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag 721 gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg 781 gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc 841 tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct 901 tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga 961 cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc 1021 gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc 1081 gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc 1141 ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc 1201 gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt 1261 cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgag 1321 cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca 1381 cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga 1441 atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag 1501 tttttttctt ccatttcagg tgtcgtgagg taccggatcc tctagagtcg actccggaat 1561 aacttcgtat aggatacttt atacgaagtt atgcagaatg gtagctggat tgtagctgct 1621 attagcaata tgaaacctct taataacttc gtatagcata cattatacga agttatggcg 1681 cgccttacag tcgggtttcg ccgcctgatt tatggctttc ccaactacta atgatcttgc 1741 ctgactgagg tgtgactgac acttctctcc cggttcctcg cagattgtgc tgagtgggct 1801 cactccggtt cacgcgccga cagcaggtca tcaggaaaat gatcagcatc agggcagtca 1861 gtgctccagc actcagcagc acgtacttgc cccaatttgg cagtcccagg tccacgccag 1921 acacctggtt atgcacatca ggcaggtgga cctccacaaa gtcttcggcc tcatctccgt 1981 ctttgaacac tgtggaggga tctgccagtg gatggaccag aggaatcacg cttgattcca 2041 gcagctccat gtgctgctgc agcagactag actgcatctc agggatcagc acgttgccgt 2101 cgggccccag aatgattcca ttaaagaaga cgccgttcac atgtgggtga cagcgtccgc 2161 ccactcgcag gcagcccttg cttggcagaa tttcattcca ggtgcgcact gatttataat 2221 gggcgtctgc ctccatcagt gtcttgttaa agatggtgta ggctttgccg aagccgggca 2281 ccagctttcg caggtgagac agcctcctga agctcacgct cttggtagtc atgatggatt 2341 ccagggcatc cagacattcc tccctttttc gcaccagttc ctcgaccacc agatgttcga 2401 tctcatcgga tctgaagtcg tgcagattca ccagtttgtc aggggggcac cacttagtct 2461 cgtttgatgt ctgcatactg acccaggtgc catccatcag tctcagtccc agcacgccac 2521 acagtttcag cttgcaagcc cctttcaggc tcttatacag tcccctttcg tccacaaacc 2581 cacaagtctc gcttcctttt gaggctcgct tgccgcggga atttgtgaaa atgtcgcagc 2641 tcatccccag cctgggattc tctggcatcc agattgtata atcatggttg gtgctacagt 2701 aagtggagct gacagccacc ccagagcact ttccggaggg aaacacccga ctgtgcagag 2761 atctatcgta tggatccagg tctgcgacag aaggggaaat gatgaccagg gactcttttg 2821 tggtcttcac agtccgcagc catctatagt ctgggtaagg attgtgcaga gattcctcat 2881 atcgtggatc gccagccatc ttccagttgt aagcggcgcg acaggcgtcg ggtgttggtc 2941 taaagtgttt cctcttgaaa gttgtggtga catagcccac aaaatttgtg taggtttctg 3001 cctcggtgac cacgccagtg catgtgaacc cgttgacctt aatagccagg atatatccca 3061 ctttcagctc catgtaggag aagccgctca ggttggtaca cccttcgtcc tcgaccacca 3121 gattgttagg gcaactcaga tggtgaatat cgataggaga ccaggggccc agtttatcag 3181 gaatagtgta gattgggaac ttgccgaagc acagtggaaa gaccagcaga gggacaaaca 3241 gcagagcctg ggggaccatg ctgccggggg cggggccggg gttctcctcc acgtcgccgc 3301 aggtcagcag gctgccgcgg ccctcaccac tacccttgtc atcgtcatcc ttgtagtcga 3361 tgtcatgatc tttataatca ccgtcatggt ctttgtagtc attaagcttg tgccccagtt 3421 tgctagggag gtcgcagtat ctggccactg ccacctcgtg ctgctcgacg taggtctcgt 3481 tgttggcctc cttgattctt tccagtctgt agtccacata gtagacgcca ggcatcttga 3541 ggttcttagc gggtttcttg gatctatatg tggtcttgat gtttgcgatc agatggctcc 3601 cgcccacgag cttcagggcc atgtcgtttc tgccttccag gccgccgtca gcggggtaca 3661 gcgtctcggt gaaggcctcc cagccgagtg ttttcttctg catcacaggg ccgttggatg 3721 tgaagttcac ccctctgatc ttgacgttgt agatgaggca gccgtcctgg aggctggtgt 3781 cctgggtagc ggtcagcacg cccccgtctt cgtatgtggt gactctctcc catgtgaagc 3841 cctcagggaa ggactgcttg aagaagtcgg ggatgccctg ggtgtggttg atgaaggtct 3901 tgctgccgta gaggaagcta gtagccagga tgtcgaaggc gaaggggaga gggccgccct 3961 cgaccacctt gattctcatg gtctgggtgc cctcgtaggg cttgccttcg ccctcggatg 4021 tgcacttgaa gtgatggttg tccacggtgc cctccatgta cagcttcatg tgcatgttct 4081 ccttaatcag ctcgctcatg gtggcgaccg gtggatcctt agcgctggtg tacttggtga 4141 tggccttagt accctcggac acggcgtgct tggccaactc cccaggcagc agcaggcgca 4201 cggccgtctg gatctccctg gaggtgatgg tcgagcgctt gttgtaatgc gccaggcggg 4261 aagcctcacc tgcgatgcgc tcgaaaatgt cgttcacaaa cgaattcatg atgcccatgg 4321 ccttggacga aatgccggtg tcagggtgga cctgcttcag aaccttgtac acatagatgg 4381 aatagctctc cttgcggctg cgcttgcgct tcttgccgcc tttcttctgc gccttagtca 4441 ccgccttctt ggagcccttt ttcggggcgg gagcagactt cgctggctct ggcatggtgg 4501 ctagcataac ttcgtataaa gtatcctata cgaagttatt tgccttaacc cagaaattat 4561 cactgttatt ctttagaatg gtgcaaagaa taacttcgta taatgtatgc tatacgaagt 4621 tatgaattcg atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga 4681 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg 4741 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc 4801 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc 4861 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt 4921 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt 4981 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg 5041 aaatcatcgt cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg 5101 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg 5161 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt 5221 tgggccgcct ccccgcatcg ataccgagcg ctgctcgaga gatctacggg tggcatccct 5281 gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc 5341 ttgtcctaat aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta 5401 tggggtggag gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct 5461 gcggggtcta ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct 5521 ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg 5581 catgcatgac caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat 5641 tggccaggct ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat 5701 tgctgggatt acaggcgtga accactgctc ccttccctgt ccttctgatt ttgtaggtaa 5761 ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct 5821 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc 5881 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta 5941 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac 6001 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 6061 acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 6121 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 6181 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca 6241 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 6301 ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa 6361 gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac 6421 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc 6481 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga 6541 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc 6601 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata 6661 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact 6721 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 6781 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 6841 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 6901 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 6961 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 7021 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 7081 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 7141 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 7201 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 7261 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 7321 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 7381 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 7441 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 7501 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 7561 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 7621 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 7681 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 7741 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 7801 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 7861 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 7921 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 7981 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta 8041 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 8101 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 8161 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 8221 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 8281 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 8341 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 8401 ca //