LOCUS       sequence-198989-        6656 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent bicistronic vector expressing mRuby2 and GCaMP6s from 
            a single open reading frame..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6656)
  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 6656)
  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-Flex-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..6656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   60..189
                     /label=AAV2 ITR
     promoter        271..718
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(734..767)
                     /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(818..851)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(866..1309,1310..1315,1316..1750,1751..1768,
                     1769..2038,2039..2044,2045..2101,2102..2128,2129..2152,
                     2153..2155,2156..2188,2189..2191,2192..2209,2210..2215))
                     /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"
     primer_bind     1425..1444
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1684..1705
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1811..1832)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2113..2122
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2216..2272)
                     /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             complement(2282..2992)
                     /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"
     regulatory      2989..2998
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3023..3056
                     /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    3107..3140
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3153..3741
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3206..3226)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3624..3635)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3744..3760)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3745..3761)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3790..3911
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3801..3820)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3855..3874
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4073..4202
                     /label=AAV2 ITR
     rep_origin      complement(4439..5027)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4519..4538)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5198..5989,5990..6058))
                     /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     5821..5840
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6059..6163)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6189..6644)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6326..6347)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6538..6557
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
       61 tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg
      121 gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta
      181 ggggttcctt gtagttaatg attaacccgc catgctactt atctacgtag ccatgctcta
      241 ggaagatctc tgcagagggc cctgcgtatg agtgcaagtg ggttttagga ccaggatgag
      301 gcggggtggg ggtgcctacc tgacgaccga ccccgaccca ctggacaagc acccaacccc
      361 cattccccaa attgcgcatc ccctatcaga gagggggagg ggaaacagga tgcggcgagg
      421 cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc ctggcggcgc
      481 gcgccaccgc cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc ccccgcaaac
      541 tccccttccc ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg gaccgcacca
      601 cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg cgctgcggcg ccggcgactc
      661 agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga gagcgcagtc
      721 gaattcagat cttataactt cgtataggat actttatacg aagttatgca gaatggtagc
      781 tggattgtag ctgctattag caatatgaaa cctcttaata acttcgtata gcatacatta
      841 tacgaagtta tactagtgcg gccgcttact tcgctgtcat catttgtaca aactcttcgt
      901 agtttacctg accatcccca tcgatgtctg cttccctgat catttcatca acctcttcat
      961 ctgttaactt ctctccaagg tttgtcatca cgtggcgaag ctctgctgca ctgatgtagc
     1021 cattgccatc cttatcaaac acaccgaacg cttctctaat ttcttcttcc gtgtccctgt
     1081 atttcatttt tcttgccatc attgtcagga actcagggaa gtcgattgtg ccgtcaccgt
     1141 cggcatctac ttcattgatc atgtcctgca gctctgcttc tgtggggttc tgccccagag
     1201 accgcatcac cgtccccagc tccttggttg ttattgtccc atccccgtcc ttgtcaaata
     1261 gggagaaagc ctctttaaat tctgcgatct gctcttcagt cagttggtcc ggcaggttgt
     1321 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
     1381 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt
     1441 cgtccttgaa gaagatggtg cgctcctgga tgtagccttc gggcatggcg gacttgaaga
     1501 agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg
     1561 tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg
     1621 tcagcttgcc gtaggtggca tcgccctcac cctcgccgga cacgctgaac ttgtggccgt
     1681 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct
     1741 tgctcaccat gctccctccg gtaccgccct tgtacagctc gtccatgccg agagtgatcc
     1801 cggcggcggt cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttcg
     1861 aaagtttgga ctgcacgctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga
     1921 tgggggtgtt ctgctggtag tggtaggcga gctgcacgcc gccgtcctcg atgttgtggc
     1981 ggatgtggaa gttcgccttg atgccgttct tctgcttgtc ggccttgata tagacgttct
     2041 cgagtgagct cagccgacct atagctctga ctgcgtgacc tgtcttattc cacttacgac
     2101 gtgatgagtc gaccatggtg gcgagatcct tatcgtcatc gtcgtacaga tcccgaccca
     2161 tttgctgtcc accagtcatg ctagccatac catgatgatg atgatgatga gaaccaggac
     2221 cggggttttc ttccacgtct cctgcttgct ttaacagaga gaagttcgtg gctccggatc
     2281 ccttgtacag ctcgtccatc ccaccaccaa gcccggcgaa cttggcaact gcgtgttcgc
     2341 gttgtactac gaacatttca ttgtcacttt cctctaacct ttccaggcgg tgatcaacgg
     2401 catggatacc gggcatcttg atgttcccga cggtcttttt tgacctgtaa gttgttacga
     2461 aagagcaaga cagatggcca ccaccatcaa ctttcagtgc catatgagtg tatcccctca
     2521 gaccaccatc tgctggatac atcatctctg tattaggctc ccaacccttg gtcttcttct
     2581 gcatcacggg accattggag ggaaagttta cccctctgac ttggacgtgg taaacgagac
     2641 agccatcctc aaggctggtg tcctgcatga cggtgacgac tccaccatct tcgtatctcg
     2701 taactctttc ccaagtaaaa ccctcaggaa aggactgttt aaagaaatca ggaatgcctt
     2761 tcgggtactt gataaaagta cggctgccat acatgaacga cgtggcaaga atgtcaaagg
     2821 caaatggcag gggtcctccc tcgatgactt tgatcctcat ggtttgagtt cccatgtacg
     2881 gattgccttc tccttcacct gtgcatttga attggtggcc gttgaccgaa ccttccatga
     2941 ccaccttcat acgcatattt tccttgatca gctcttcgcc cttagacacc atggtggccc
     3001 taggaccggt actagtctcg agataacttc gtataaagta tcctatacga agttatttgc
     3061 cttaacccag aaattatcac tgttattctt tagaatggtg caaagaataa cttcgtataa
     3121 tgtatgctat acgaagttat aagcttatcg ataatcaacc tctggattac aaaatttgtg
     3181 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     3241 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     3301 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     3361 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     3421 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     3481 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     3541 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
     3601 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     3661 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     3721 ccctttgggc cgcctccccg catcgatacc gtcgacctcg acccgggcgg ccgcttcgag
     3781 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     3841 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     3901 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt
     3961 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatcttc
     4021 ctagagcatg gctacgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc
     4081 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     4141 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     4201 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4261 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4321 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4381 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4441 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4501 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4561 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4621 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4681 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4741 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4801 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4861 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4921 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4981 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5041 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5101 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5161 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5221 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5281 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5341 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5401 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5461 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5521 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5581 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5641 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5701 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5761 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5821 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5881 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5941 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6001 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6061 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6121 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6181 tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     6241 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     6301 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     6361 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     6421 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     6481 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     6541 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     6601 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccat
//
