LOCUS       sequence-120856-        7112 bp    DNA     circular SYN 14-APR-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 7112)
  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 7112)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-CAG-Flex-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..7112
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        251..528
                     /label=chicken beta-actin promoter
     primer_bind     808..830
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     892..911
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(945..978)
                     /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(1029..1062)
                     /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(1077..1520,1521..1526,1527..1961,1962..1979,
                     1980..2249,2250..2255,2256..2312,2313..2339,2340..2363,
                     2364..2366,2367..2399,2400..2402,2403..2420,2421..2426))
                     /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     1636..1655
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1895..1916
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2022..2043)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2324..2333
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2427..2483)
                     /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(2493..3203)
                     /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      3200..3209
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3234..3267
                     /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    3318..3351
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3364..3952
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3417..3437)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3835..3846)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3995..4116
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4006..4025)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4060..4079
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4278..4407
                     /label=AAV2 ITR
     rep_origin      complement(4644..5232)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4724..4743)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5403..6194,6195..6263))
                     /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     6026..6045
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6264..6368)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6394..6849)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6531..6552)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6743..6762
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6921..7050
                     /label=AAV2 ITR
ORIGIN
        1 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
       61 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      121 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      181 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      241 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc
      301 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg
      361 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg
      421 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
      481 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
      541 acgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
      601 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
      661 ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg
      721 gctccgggag ggccctttgt gcggggggag cggctcgggg ctgtccgcgg ggggacggct
      781 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
      841 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
      901 tggttattgt gctgtctcat cattttggca aagaattgga tcttataact tcgtatagga
      961 tactttatac gaagttatgc agaatggtag ctggattgta gctgctatta gcaatatgaa
     1021 acctcttaat aacttcgtat agcatacatt atacgaagtt atactagtgc ggccgcttac
     1081 ttcgctgtca tcatttgtac aaactcttcg tagtttacct gaccatcccc atcgatgtct
     1141 gcttccctga tcatttcatc aacctcttca tctgttaact tctctccaag gtttgtcatc
     1201 acgtggcgaa gctctgctgc actgatgtag ccattgccat ccttatcaaa cacaccgaac
     1261 gcttctctaa tttcttcttc cgtgtccctg tatttcattt ttcttgccat cattgtcagg
     1321 aactcaggga agtcgattgt gccgtcaccg tcggcatcta cttcattgat catgtcctgc
     1381 agctctgctt ctgtggggtt ctgccccaga gaccgcatca ccgtccccag ctccttggtt
     1441 gttattgtcc catccccgtc cttgtcaaat agggagaaag cctctttaaa ttctgcgatc
     1501 tgctcttcag tcagttggtc cggcaggttg tactccagct tgtgccccag gatgttgccg
     1561 tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac
     1621 ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg
     1681 atgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag
     1741 cggctgaagc actgcacgcc gtaggtcagg gtggtcacga gggtgggcca gggcacgggc
     1801 agcttgccgg tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc atcgccctca
     1861 ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccagctc gaccaggatg
     1921 ggcaccaccc cggtgaacag ctcctcgccc ttgctcacca tgctccctcc ggtaccgccc
     1981 ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc cagcaggacc
     2041 atgtgatcgc gcttctcgtt ggggtctttc gaaagtttgg actgcacgct caggtagtgg
     2101 ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtaggcg
     2161 agctgcacgc cgccgtcctc gatgttgtgg cggatgtgga agttcgcctt gatgccgttc
     2221 ttctgcttgt cggccttgat atagacgttc tcgagtgagc tcagccgacc tatagctctg
     2281 actgcgtgac ctgtcttatt ccacttacga cgtgatgagt cgaccatggt ggcgagatcc
     2341 ttatcgtcat cgtcgtacag atcccgaccc atttgctgtc caccagtcat gctagccata
     2401 ccatgatgat gatgatgatg agaaccagga ccggggtttt cttccacgtc tcctgcttgc
     2461 tttaacagag agaagttcgt ggctccggat cccttgtaca gctcgtccat cccaccacca
     2521 agcccggcga acttggcaac tgcgtgttcg cgttgtacta cgaacatttc attgtcactt
     2581 tcctctaacc tttccaggcg gtgatcaacg gcatggatac cgggcatctt gatgttcccg
     2641 acggtctttt ttgacctgta agttgttacg aaagagcaag acagatggcc accaccatca
     2701 actttcagtg ccatatgagt gtatcccctc agaccaccat ctgctggata catcatctct
     2761 gtattaggct cccaaccctt ggtcttcttc tgcatcacgg gaccattgga gggaaagttt
     2821 acccctctga cttggacgtg gtaaacgaga cagccatcct caaggctggt gtcctgcatg
     2881 acggtgacga ctccaccatc ttcgtatctc gtaactcttt cccaagtaaa accctcagga
     2941 aaggactgtt taaagaaatc aggaatgcct ttcgggtact tgataaaagt acggctgcca
     3001 tacatgaacg acgtggcaag aatgtcaaag gcaaatggca ggggtcctcc ctcgatgact
     3061 ttgatcctca tggtttgagt tcccatgtac ggattgcctt ctccttcacc tgtgcatttg
     3121 aattggtggc cgttgaccga accttccatg accaccttca tacgcatatt ttccttgatc
     3181 agctcttcgc ccttagacac catggtggcc ctaggaccgg tactagtctc gagataactt
     3241 cgtataaagt atcctatacg aagttatttg ccttaaccca gaaattatca ctgttattct
     3301 ttagaatggt gcaaagaata acttcgtata atgtatgcta tacgaagtta taagcttatc
     3361 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     3421 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     3481 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     3541 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     3601 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     3661 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     3721 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     3781 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     3841 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     3901 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
     3961 cgtcgacccg ggcggccgct tcgagcagac atgataagat acattgatga gtttggacaa
     4021 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     4081 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     4141 atgtttcagg ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa
     4201 tgtggtaaaa tcgataagga tcttcctaga gcatggctac gtagataagt agcatggcgg
     4261 gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4321 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     4381 ggcctcagtg agcgagcgag cgcgcagctg cattaatgaa tcggccaacg cgcggggaga
     4441 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4501 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4561 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4621 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4681 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4741 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4801 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4861 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4921 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4981 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5041 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5101 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5161 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5221 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5281 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5341 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5401 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5461 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5521 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5581 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5641 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5701 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5761 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5821 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5881 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5941 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6001 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6061 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6121 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6181 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6241 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6301 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6361 gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa
     6421 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     6481 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     6541 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     6601 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     6661 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     6721 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     6781 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     6841 agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     6901 cctcttcgct attacgccag ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc
     6961 ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg
     7021 gagtggccaa ctccatcact aggggttcct tgtagttaat gattaacccg ccatgctact
     7081 tatctacgta gccatgctct aggaagatcg ta
//
