LOCUS       Exported                6414 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Bicistronic vector expressing mRuby2 and GCaMP6s from a single open 
            reading frame..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-hSyn1-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6414)
  AUTHORS   Rose T, Jaepel J, Hubener M, Bonhoeffer T
  TITLE     Cell-specific restoration of stimulus preference after monocular 
            deprivation in the visual cortex.
  JOURNAL   Science. 2016 Jun 10;352(6291):1319-22. doi: 
            10.1126/science.aad3358.
  PUBMED    27284193
REFERENCE   2  (bases 1 to 6414)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6414
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     regulatory      130..139
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             136..846
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     CDS             856..912
                     /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             913..2262
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="GSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKT
                     GHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPV
                     LLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEEL
                     FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
                     GVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL
                     KGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSL
                     GQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGNG
                     YISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1006..1015
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     1296..1317
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1423..1444)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1684..1703)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2287..2875
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2340..2360)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2758..2769)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2878..2894)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2879..2895)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    2924..3045
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2935..2954)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2989..3008
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3207..3336
                     /label=AAV2 ITR
     primer_bind     complement(3402..3419)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3573..4161)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3653..3672)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4332..5192)
                     /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     4955..4974
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5193..5297)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5323..5778)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5460..5481)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5672..5691
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5850..5979
                     /label=AAV2 ITR
     promoter        6061..94
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
ORIGIN
        1 cgggcgcgac catctgcgct gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg
       61 cagcggagga gtcgtgtcgt gcctgagagc gcagtcgaat tcaagctgct agctagtacc
      121 ggtcctaggg ccaccatggt gtctaagggc gaagagctga tcaaggaaaa tatgcgtatg
      181 aaggtggtca tggaaggttc ggtcaacggc caccaattca aatgcacagg tgaaggagaa
      241 ggcaatccgt acatgggaac tcaaaccatg aggatcaaag tcatcgaggg aggacccctg
      301 ccatttgcct ttgacattct tgccacgtcg ttcatgtatg gcagccgtac ttttatcaag
      361 tacccgaaag gcattcctga tttctttaaa cagtcctttc ctgagggttt tacttgggaa
      421 agagttacga gatacgaaga tggtggagtc gtcaccgtca tgcaggacac cagccttgag
      481 gatggctgtc tcgtttacca cgtccaagtc agaggggtaa actttccctc caatggtccc
      541 gtgatgcaga agaagaccaa gggttgggag cctaatacag agatgatgta tccagcagat
      601 ggtggtctga ggggatacac tcatatggca ctgaaagttg atggtggtgg ccatctgtct
      661 tgctctttcg taacaactta caggtcaaaa aagaccgtcg ggaacatcaa gatgcccggt
      721 atccatgccg ttgatcaccg cctggaaagg ttagaggaaa gtgacaatga aatgttcgta
      781 gtacaacgcg aacacgcagt tgccaagttc gccgggcttg gtggtgggat ggacgagctg
      841 tacaagggat ccggagccac gaacttctct ctgttaaagc aagcaggaga cgtggaagaa
      901 aaccccggtc ctggttctca tcatcatcat catcatggta tggctagcat gactggtgga
      961 cagcaaatgg gtcgggatct gtacgacgat gacgataagg atctcgccac catggtcgac
     1021 tcatcacgtc gtaagtggaa taagacaggt cacgcagtca gagctatagg tcggctgagc
     1081 tcactcgaga acgtctatat caaggccgac aagcagaaga acggcatcaa ggcgaacttc
     1141 cacatccgcc acaacatcga ggacggcggc gtgcagctcg cctaccacta ccagcagaac
     1201 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cgtgcagtcc
     1261 aaactttcga aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1321 gccgccggga tcactctcgg catggacgag ctgtacaagg gcggtaccgg agggagcatg
     1381 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1441 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggtg agggcgatgc cacctacggc
     1501 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1561 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     1621 cacgacttct tcaagtccgc catgcccgaa ggctacatcc aggagcgcac catcttcttc
     1681 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1741 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1801 ctggagtaca acctgccgga ccaactgact gaagagcaga tcgcagaatt taaagaggct
     1861 ttctccctat ttgacaagga cggggatggg acaataacaa ccaaggagct ggggacggtg
     1921 atgcggtctc tggggcagaa ccccacagaa gcagagctgc aggacatgat caatgaagta
     1981 gatgccgacg gtgacggcac aatcgacttc cctgagttcc tgacaatgat ggcaagaaaa
     2041 atgaaataca gggacacgga agaagaaatt agagaagcgt tcggtgtgtt tgataaggat
     2101 ggcaatggct acatcagtgc agcagagctt cgccacgtga tgacaaacct tggagagaag
     2161 ttaacagatg aagaggttga tgaaatgatc agggaagcag acatcgatgg ggatggtcag
     2221 gtaaactacg aagagtttgt acaaatgatg acagcgaagt aagcggccgc actagagctt
     2281 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     2341 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     2401 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     2461 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     2521 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     2581 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     2641 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     2701 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     2761 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     2821 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     2881 taccgtcgac ctcgacccgg gcggccgctt cgagcagaca tgataagata cattgatgag
     2941 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     3001 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     3061 attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac
     3121 ctctacaaat gtggtaaaat cgataaggat cttcctagag catggctacg tagataagta
     3181 gcatggcggg ttaatcatta actacaagga acccctagtg atggagttgg ccactccctc
     3241 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     3301 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc attaatgaat cggccaacgc
     3361 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3421 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3481 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3541 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3601 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3661 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3721 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3781 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3841 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3901 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3961 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4021 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4081 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4141 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4201 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4261 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4321 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4381 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4441 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4501 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4561 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4621 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4681 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4741 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4801 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4861 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4921 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4981 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5041 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5101 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5161 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5221 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5281 caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata
     5341 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     5401 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5461 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5521 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5581 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5641 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5701 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     5761 cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     5821 cggtgcgggc ctcttcgcta ttacgccagc tgcgcgctcg ctcgctcact gaggccgccc
     5881 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc
     5941 gcagagaggg agtggccaac tccatcacta ggggttcctt gtagttaatg attaacccgc
     6001 catgctactt atctacgtag ccatgctcta ggaagatctc tgcagagggc cctgcgtatg
     6061 agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga
     6121 ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga
     6181 gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg
     6241 gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc
     6301 gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg
     6361 cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggca
//
