LOCUS       Exported                7370 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  mGFP and Synaptophysin-mRuby expression from Cre-expressing neurons.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV hSyn FLEx mGFP-2A-Synaptophysin-mRuby
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7370)
  AUTHORS   Beier KT, Steinberg EE, DeLoach KE, Xie S, Miyamichi K, Schwarz L, 
            Gao XJ, Kremer EJ, Malenka RC, Luo L
  TITLE     Circuit Architecture of VTA Dopamine Neurons Revealed by Systematic 
            Input-Output Mapping.
  JOURNAL   Cell. 2015 Jul 30;162(3):622-34. doi: 10.1016/j.cell.2015.07.015.
  PUBMED    26232228
REFERENCE   2  (bases 1 to 7370)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7370
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     polyA_signal    1..477
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     259..278
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    509..1097
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             615..626
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1024..1044
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(1145..1178)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(1249..1282)
                     /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)."
     CDS             1355..2071
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1355..2071
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1400..1421)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1661..1680)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2008..2029
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2072..2125
                     /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             3077..3757
                     /codon_start=1
                     /product="monomeric variant of the red fluorescent protein 
                     eqFP611 (Kredel et al., 2009)"
                     /label=mRuby
                     /translation="MNSLIKENMRMKVVLEGSVNGHQFKCTGEGEGNPYMGTQTMRIKV
                     IEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGVITV
                     MQDTSLEDGCLVYHAQVRGVNFPSNGAVMQKKTKGWEPNTEMMYPADGGLRGYTHMALK
                     VDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVAKFA
                     GLGGG"
     protein_bind    3767..3800
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    3863..3896
                     /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)."
     promoter        complement(3972..4419)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     repeat_region   4464..4593
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4655..5243)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4735..4754)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5414..6274)
                     /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     6037..6056
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6275..6379)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6447..6465
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6503..6525)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6625..6644
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6661..7116)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6798..6819)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7010..7029
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   7191..7331
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7202..7331
                     /label=AAV2 ITR
ORIGIN
        1 aaggacaggg aagggagcag tggttcacgc ctgtaatccc agcaatttgg gaggccaagg
       61 tgggtagatc acctgagatt aggagttgga gaccagcctg gccaatatgg tgaaaccccg
      121 tctctaccaa aaaaacaaaa attagctgag cctggtcatg catgcctgga atcccaacaa
      181 ctcgggaggc tgaggcagga gaatcgcttg aacccaggag gcggagattg cagtgagcca
      241 agattgtgcc actgcactcc agcttggttc ccaatagacc ccgcaggccc tacaggttgt
      301 cttcccaact tgccccttgc tccataccac ccccctccac cccataatat tatagaagga
      361 cacctagtca gacaaaatga tgcaacttaa ttttattagg acaaggctgg tgggcactgg
      421 agtggcaact tccagggcca ggagaggcac tggggagggg tcacagggat gccacccgta
      481 gatctctcga gcagcgctcg gtatcgatgc ggggaggcgg cccaaaggga gatccgactc
      541 gtctgagggc gaaggcgaag acgcggaaga ggccgcagag ccggcagcag gccgcgggaa
      601 ggaaggtccg ctggattgag ggccgaaggg acgtagcaga aggacgtccc gcgcagaatc
      661 caggtggcaa cataggcgag cagccaagga aaggacgatg atttccccga caacaccacg
      721 gaattgtcag tgcccaacag ccgagcccct gtccagcagc gggcaaggca ggcggcgatg
      781 agttccgccg tggcaatagg gagggggaaa gcgaaagtcc cggaaaggag ctgacaggtg
      841 gtggcaatgc cccaaccagt gggggttgcg tcagcaaaca cagtgcacac cacgccacgt
      901 tgcctgacaa cgggccacaa ctcctcataa agagacagca accaggattt atacaaggag
      961 gagaaaatga aagccatacg ggaagcaata gcatgataca aaggcattaa agcagcgtat
     1021 ccacatagcg taaaaggagc aacatagtta agaataccag tcaatctttc acaaattttg
     1081 taatccagag gttgattatc gataagcttg atatcgaatt catcgatcgg ccgcatataa
     1141 gcttataact tcgtataatg tatgctatac gaagttatgg gtcgatggtg agatctggac
     1201 tagagggtcg atggtgatgc ttggatcaaa ataggaagaa tcccaatgat aacttcgtat
     1261 aaagtatcct atacgaagtt ataggcgcgc caccatgctg tgctgtatga gaagaaccaa
     1321 acaggttgaa aagaatgatg aggaccaaaa gatcatggtg agcaagggcg aggagctgtt
     1381 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     1441 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     1501 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     1561 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     1621 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     1681 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     1741 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     1801 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     1861 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     1921 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     1981 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     2041 gatcactctc ggcatggacg agctgtacaa ggagggcagg ggcagcctgc tgacctgcgg
     2101 cgacgtggag gagaaccccg gccccatgga cgtggtgaat cagctggtgg ctgggggtca
     2161 gttccgggtg gtcaaggagc cccttggctt cgtgaaggtg ctgcagtggg tctttgccat
     2221 cttcgccttt gctacgtgcg gcagctacac cggagagctt cggctgagcg tggagtgtgc
     2281 caacaagacg gagagtgccc tcaacatcga agtcgaattt gagtacccat tcaggctgca
     2341 ccaagtgtac tttgatgcac cctcctgcgt taaagggggc actaccaaga tcttcctagt
     2401 tggtgactac tcctcctcgg ctgaattctt tgtcaccgtg gctgtgtttg ccttcctcta
     2461 ctccatgggg gccctggcca cctacatctt cctgcagaac aagtaccgag agaacaacaa
     2521 agggccaatg atggacttcc tggccacagc agtgttcgct ttcatgtggc tagttagctc
     2581 atccgcctgg gccaaaggcc tgtccgatgt gaagatggcc actgacccag agaacattat
     2641 caaggagatg cctatgtgcc gccagacagg aaacacatgc aaggaactga gggaccctgt
     2701 gacttcagga ctcaacacct cggtggtgtt tggcttcctg aacctggtgc tctgggttgg
     2761 caacctatgg ttcgtgttca aggagacagg ctgggccgcc ccattcatgc gcgcacctcc
     2821 aggcgcccca gaaaagcaac cagctcctgg cgatgcctac ggcgatgcgg gctatgggca
     2881 gggccccgga ggctatgggc cccaggactc ctacgggcct cagggtggtt atcaacccga
     2941 ttacgggcag ccagccagcg gcggtggcgg tggctacggg cctcagggcg actatgggca
     3001 gcaaggctac ggccaacagg gtgcgcccac ctccttctcc aatcagatgt cggatccacc
     3061 ggtcgccacc cgtacgatga acagcctgat caaagaaaac atgcggatga aggtggtgct
     3121 ggaaggcagc gtgaacggcc accagttcaa gtgcaccggc gagggcgagg gcaaccccta
     3181 catgggcacc cagaccatgc ggatcaaagt gatcgagggc ggacctctgc ccttcgcctt
     3241 cgacatcctg gccacatcct tcatgtacgg cagccggacc ttcatcaagt accccaaggg
     3301 catccccgat ttcttcaagc agagcttccc cgagggcttc acctgggaga gagtgaccag
     3361 atacgaggac ggcggcgtga tcaccgtgat gcaggacacc agcctggaag atggctgcct
     3421 ggtgtaccat gcccaggtca ggggcgtgaa ttttcccagc aacggcgccg tgatgcagaa
     3481 gaaaaccaag ggctgggagc ccaacaccga gatgatgtac cccgctgacg gcggactgag
     3541 aggctacacc cacatggccc tgaaggtgga cggcggaggg cacctgagct gcagcttcgt
     3601 gaccacctac cgatccaaga aaaccgtggg caacatcaag atgcccggca tccacgccgt
     3661 ggaccaccgg ctggaaaggc tggaagagtc cgacaacgag atgttcgtgg tgcagcggga
     3721 gcacgccgtg gccaagttcg ccggcctggg cggagggtaa gtcgacataa cttcgtatag
     3781 catacattat acgaagttat gtgtcgatgg tgatgcttgg caattcgggt cgatggtgaa
     3841 gcattggtct tcctattttg atataacttc gtataggata ctttatacga agttatcccg
     3901 gccgggcccg cggtatcaaa tcaattcttt gccaaaatga tgagacagca caggatccgg
     3961 taccttctcg actgcgctct caggcacgac acgactcctc cgctgcccac cgcagactga
     4021 ggcagcgctg agtcgccggc gccgcagcgc agatggtcgc gcccgtgccc ccctatctcg
     4081 cgcctcgcgt ggtgcggtcc ggctgggccg gcggcggcgc ggacgcgacc aaggtggccg
     4141 ggaaggggag tttgcggggg accggcgagt gacgtcagcg cgccttcagt gctgaggcgg
     4201 cggtggcgcg cgccgccagg cgggggcgaa ggcactgtcc gcggtgctga agctggcagt
     4261 gcgcacgcgc ctcgccgcat cctgtttccc ctccccctct ctgatagggg atgcgcaatt
     4321 tggggaatgg gggttgggtg cttgtccagt gggtcggggt cggtcgtcag gtaggcaccc
     4381 ccaccccgcc tcatcctggt cctaaaaccc acttgcactc atacgcaggg ccctctgcag
     4441 tctagacaca cgcgtgcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     4501 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct
     4561 cagtgagcga gcgagcgcgc agctgcctgc aggacatgtg agcaaaaggc cagcaaaagg
     4621 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4681 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4741 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4801 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4861 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4921 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4981 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5041 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5101 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5161 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5221 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5281 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5341 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5401 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5461 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5521 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5581 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5641 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5701 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5761 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5821 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5881 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5941 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6001 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6061 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6121 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6181 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6241 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6301 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6361 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     6421 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc
     6481 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     6541 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     6601 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     6661 aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     6721 tttttaacca atagaccgaa atcggcaaaa tcccttataa atcaaaagaa tagcccgaga
     6781 tagagttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     6841 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccca
     6901 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     6961 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     7021 cgaaaggagc gggcgctaag gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     7081 cacccgccgc gcttaatgcg ccgctacagg gcgcgtacta tggttgcttt gacgtatgcg
     7141 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc cctgcaggca
     7201 gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt
     7261 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
     7321 taggggttcc tgcggccgct cggtccgcac gtggttacct acaaaatcag
//
