LOCUS       sequence-108519-        6722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bicistronic vector expressing mRuby2 and GCaMP6f from a single open 
            reading frame. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6722)
  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 6722)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2016-06-10- 10"; volume: "352"; 
            issue: "6291"; pages: "1319-22"
COMMENT     Sequence Label: pAAV-hSyn1-mRuby2-GSG-P2A-GCaMP6f-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..6722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        22..469
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      505..514
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             511..1221
                     /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             1231..1287
                     /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             join(1288..1293,1294..1311,1312..1314,1315..1347,1348..1350,
                     1351..1374,1375..1401,1402..1458,1459..1464,1465..1734,
                     1735..1752,1753..2187,2188..2193,2194..2637)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="GSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKT
                     GHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPV
                     LLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEEL
                     FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
                     GVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL
                     KGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRSL
                     GQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGNG
                     YISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1381..1390
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1671..1692
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1798..1819)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2059..2078)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2662..3250
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2715..2735)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3133..3144)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3253..3269)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3254..3270)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3299..3420
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3310..3329)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3364..3383
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3582..3711
                     /label=AAV2 ITR
     primer_bind     complement(3730..3747)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3730..3746)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3739..3761)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3888..4343
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3975..3994)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4185..4206
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4369..4473
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4474..4542,4543..5334)
                     /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(4692..4711)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5505..6093
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5994..6013
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6381..6402
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6417..6422,6423..6440,6441..6447)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6455..6471
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     6460..6482
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6479..6495
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6479..6495
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   6533..6662
                     /label=AAV2 ITR
ORIGIN
        1 ctgcagaggg ccctgcgtat gagtgcaagt gggttttagg accaggatga ggcggggtgg
       61 gggtgcctac ctgacgaccg accccgaccc actggacaag cacccaaccc ccattcccca
      121 aattgcgcat cccctatcag agagggggag gggaaacagg atgcggcgag gcgcgtgcgc
      181 actgccagct tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg cgcgccaccg
      241 ccgcctcagc actgaaggcg cgctgacgtc actcgccggt cccccgcaaa ctccccttcc
      301 cggccacctt ggtcgcgtcc gcgccgccgc cggcccagcc ggaccgcacc acgcgaggcg
      361 cgagataggg gggcacgggc gcgaccatct gcgctgcggc gccggcgact cagcgctgcc
      421 tcagtctgcg gtgggcagcg gaggagtcgt gtcgtgcctg agagcgcagt cgaattcaag
      481 ctgctagcta gtaccggtcc tagggccacc atggtgtcta agggcgaaga gctgatcaag
      541 gaaaatatgc gtatgaaggt ggtcatggaa ggttcggtca acggccacca attcaaatgc
      601 acaggtgaag gagaaggcaa tccgtacatg ggaactcaaa ccatgaggat caaagtcatc
      661 gagggaggac ccctgccatt tgcctttgac attcttgcca cgtcgttcat gtatggcagc
      721 cgtactttta tcaagtaccc gaaaggcatt cctgatttct ttaaacagtc ctttcctgag
      781 ggttttactt gggaaagagt tacgagatac gaagatggtg gagtcgtcac cgtcatgcag
      841 gacaccagcc ttgaggatgg ctgtctcgtt taccacgtcc aagtcagagg ggtaaacttt
      901 ccctccaatg gtcccgtgat gcagaagaag accaagggtt gggagcctaa tacagagatg
      961 atgtatccag cagatggtgg tctgagggga tacactcata tggcactgaa agttgatggt
     1021 ggtggccatc tgtcttgctc tttcgtaaca acttacaggt caaaaaagac cgtcgggaac
     1081 atcaagatgc ccggtatcca tgccgttgat caccgcctgg aaaggttaga ggaaagtgac
     1141 aatgaaatgt tcgtagtaca acgcgaacac gcagttgcca agttcgccgg gcttggtggt
     1201 gggatggacg agctgtacaa gggatccgga gccacgaact tctctctgtt aaagcaagca
     1261 ggagacgtgg aagaaaaccc cggtcctggt tctcatcatc atcatcatca tggtatggct
     1321 agcatgactg gtggacagca aatgggtcgg gatctgtacg acgatgacga taaggatctc
     1381 gccaccatgg tcgactcatc acgtcgtaag tggaataaga caggtcacgc agtcagagct
     1441 ataggtcggc tgagctcact cgagaacgtc tatatcaagg ccgacaagca gaagaacggc
     1501 atcaaggcga acttcaagat ccgccacaac atcgaggacg gcggcgtgca gctcgcctac
     1561 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1621 ctgagcgtgc agtccaaact ttcgaaagac cccaacgaga agcgcgatca catggtcctg
     1681 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggcggt
     1741 accggaggga gcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1801 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggtgagggc
     1861 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1921 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     1981 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta catccaggag
     2041 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     2101 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     2161 atcctggggc acaagctgga gtacaacctg ccggaccaac tgactgaaga gcagatcgca
     2221 gaatttaaag aggaattctc cctatttgac aaggacgggg atgggacaat aacaaccaag
     2281 gagctgggga cggtgatgcg gtctctgggg cagaacccca cagaagcaga gctgcaggac
     2341 atgatcaatg aagtagatgc cgacggtgac ggcacaatcg acttccctga gttcctgaca
     2401 atgatggcaa gaaaaatgaa atacagggac acggaagaag aaattagaga agcgttcggt
     2461 gtgtttgata aggatggcaa tggctacatc agtgcagcag agcttcgcca cgtgatgaca
     2521 aaccttggag agaagttaac agatgaagag gttgatgaaa tgatcaggga agcagacatc
     2581 gatggggatg gtcaggtaaa ctacgaagag tttgtacaaa tgatgacagc gaagtaagcg
     2641 gccgcactag agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     2701 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     2761 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     2821 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     2881 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     2941 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     3001 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     3061 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     3121 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     3181 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     3241 gcctccccgc atcgataccg tcgacctcga cccgggcggc cgcttcgagc agacatgata
     3301 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     3361 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     3421 aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg ggaggttttt
     3481 taaagcaagt aaaacctcta caaatgtggt aaaatcgata aggatcttcc tagagcatgg
     3541 ctacgtagat aagtagcatg gcgggttaat cattaactac aaggaacccc tagtgatgga
     3601 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     3661 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gccttaatta
     3721 acctaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     3781 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     3841 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
     3901 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3961 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4021 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4081 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     4141 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4201 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     4261 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4321 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     4381 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4441 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     4501 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     4561 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     4621 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     4681 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     4741 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     4801 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     4861 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     4921 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     4981 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5041 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5101 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     5161 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     5221 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     5281 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     5341 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5401 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     5461 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     5521 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     5581 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     5641 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     5701 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5761 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5821 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5881 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5941 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6001 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6061 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6121 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     6181 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     6241 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     6301 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     6361 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     6421 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     6481 ggaaacagct atgaccatga ttacgccaga tttaattaag gccttaatta ggctgcgcgc
     6541 tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc
     6601 ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc
     6661 cttgtagtta atgattaacc cgccatgcta cttatctacg tagccatgct ctaggaagat
     6721 ct
//
