LOCUS       sequence-198991-        7106 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Cre-dependent bicistronic vector expressing mRuby2 and GCaMP6s from 
            a single open reading frame..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7106)
  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 7106)
  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-CAG-Flex-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..7106
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     repeat_region   430..559
                     /label=AAV2 ITR
     rep_origin      631..1086
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(718..737)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     928..949
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1112..1216
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1217..1285,1286..2077)
                     /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(1435..1454)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2248..2836
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2737..2756
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   3073..3202
                     /label=AAV2 ITR
     polyA_signal    3364..3485
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3401..3420)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3455..3474
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    3528..4116
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3634..3645
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     4043..4063
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(4129..4162)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(4213..4246)
                     /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)."
     regulatory      4271..4280
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4277..4987
                     /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             4997..5053
                     /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(5054..5059,5060..5077,5078..5080,5081..5113,5114..5116,
                     5117..5140,5141..5167,5168..5224,5225..5230,5231..5500,
                     5501..5518,5519..5953,5954..5959,5960..6403)
                     /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      5147..5156
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     5437..5458
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5564..5585)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5825..5844)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    6418..6451
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    6502..6535
                     /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)."
     primer_bind     complement(6569..6588)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(6650..6672)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
ORIGIN
        1 cccccccccc cccgccccca tcgctgcaca aaataattaa aaaataaata aatacaaaat
       61 tgggggtggg gagggggggg agatggggag agtgaagcag aacgtggggc tcacctcgac
      121 catggtaata gcgatgacta atacgtagat gtactgccaa gtaggaaagt cccataaggt
      181 catgtactgg gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt
      241 acttggcata tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac
      301 ccattgacgt caatggaaag tccctattgg cgttactatg ggaacatacg tcattattga
      361 cgtcaatggt actcttccta gagcatggct acgtagataa gtagcatggc gggttaatca
      421 ttaactacaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
      481 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
      541 tgagcgagcg agcgcgcagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
      601 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
      661 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
      721 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
      781 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
      841 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
      901 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
      961 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     1021 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     1081 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1141 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     1201 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     1261 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     1321 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     1381 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     1441 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     1501 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     1561 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     1621 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     1681 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     1741 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     1801 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     1861 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     1921 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     1981 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     2041 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     2101 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     2161 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2221 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2281 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2341 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     2401 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2461 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2521 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2581 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2641 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     2701 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     2761 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     2821 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     2881 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     2941 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3001 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     3061 tcattaatgc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt
     3121 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
     3181 aactccatca ctaggggttc cttgtagtta atgattaacc cgccatgcta cttatctacg
     3241 tagccatgct ctaggaagat ccttatcgat tttaccacat ttgtagaggt tttacttgct
     3301 ttaaaaaacc tcccacatct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     3361 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     3421 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     3481 tcttatcatg tctgctcgaa gcggccgccc gggtcgacgg tatcgatgcg gggaggcggc
     3541 ccaaagggag atccgactcg tctgagggcg aaggcgaaga cgcggaagag gccgcagagc
     3601 cggcagcagg ccgcgggaag gaaggtccgc tggattgagg gccgaaggga cgtagcagaa
     3661 ggacgtcccg cgcagaatcc aggtggcaac acaggcgagc agccaaggaa aggacgatga
     3721 tttccccgac aacaccacgg aattgtcagt gcccaacagc cgagcccctg tccagcagcg
     3781 ggcaaggcag gcggcgatga gttccgccgt ggcaataggg agggggaaag cgaaagtccc
     3841 ggaaaggagc tgacaggtgg tggcaatgcc ccaaccagtg ggggttgcgt cagcaaacac
     3901 agtgcacacc acgccacgtt gcctgacaac gggccacaac tcctcataaa gagacagcaa
     3961 ccaggattta tacaaggagg agaaaatgaa agccatacgg gaagcaatag catgatacaa
     4021 aggcattaaa gcagcgtatc cacatagcgt aaaaggagca acatagttaa gaataccagt
     4081 caatctttca caaattttgt aatccagagg ttgattatcg ataagcttat aacttcgtat
     4141 agcatacatt atacgaagtt attctttgca ccattctaaa gaataacagt gataatttct
     4201 gggttaaggc aaataacttc gtataggata ctttatacga agttatctcg agactagtac
     4261 cggtcctagg gccaccatgg tgtctaaggg cgaagagctg atcaaggaaa atatgcgtat
     4321 gaaggtggtc atggaaggtt cggtcaacgg ccaccaattc aaatgcacag gtgaaggaga
     4381 aggcaatccg tacatgggaa ctcaaaccat gaggatcaaa gtcatcgagg gaggacccct
     4441 gccatttgcc tttgacattc ttgccacgtc gttcatgtat ggcagccgta cttttatcaa
     4501 gtacccgaaa ggcattcctg atttctttaa acagtccttt cctgagggtt ttacttggga
     4561 aagagttacg agatacgaag atggtggagt cgtcaccgtc atgcaggaca ccagccttga
     4621 ggatggctgt ctcgtttacc acgtccaagt cagaggggta aactttccct ccaatggtcc
     4681 cgtgatgcag aagaagacca agggttggga gcctaataca gagatgatgt atccagcaga
     4741 tggtggtctg aggggataca ctcatatggc actgaaagtt gatggtggtg gccatctgtc
     4801 ttgctctttc gtaacaactt acaggtcaaa aaagaccgtc gggaacatca agatgcccgg
     4861 tatccatgcc gttgatcacc gcctggaaag gttagaggaa agtgacaatg aaatgttcgt
     4921 agtacaacgc gaacacgcag ttgccaagtt cgccgggctt ggtggtggga tggacgagct
     4981 gtacaaggga tccggagcca cgaacttctc tctgttaaag caagcaggag acgtggaaga
     5041 aaaccccggt cctggttctc atcatcatca tcatcatggt atggctagca tgactggtgg
     5101 acagcaaatg ggtcgggatc tgtacgacga tgacgataag gatctcgcca ccatggtcga
     5161 ctcatcacgt cgtaagtgga ataagacagg tcacgcagtc agagctatag gtcggctgag
     5221 ctcactcgag aacgtctata tcaaggccga caagcagaag aacggcatca aggcgaactt
     5281 ccacatccgc cacaacatcg aggacggcgg cgtgcagctc gcctaccact accagcagaa
     5341 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcgtgcagtc
     5401 caaactttcg aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     5461 cgccgccggg atcactctcg gcatggacga gctgtacaag ggcggtaccg gagggagcat
     5521 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     5581 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggt gagggcgatg ccacctacgg
     5641 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     5701 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     5761 gcacgacttc ttcaagtccg ccatgcccga aggctacatc caggagcgca ccatcttctt
     5821 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     5881 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     5941 gctggagtac aacctgccgg accaactgac tgaagagcag atcgcagaat ttaaagaggc
     6001 tttctcccta tttgacaagg acggggatgg gacaataaca accaaggagc tggggacggt
     6061 gatgcggtct ctggggcaga accccacaga agcagagctg caggacatga tcaatgaagt
     6121 agatgccgac ggtgacggca caatcgactt ccctgagttc ctgacaatga tggcaagaaa
     6181 aatgaaatac agggacacgg aagaagaaat tagagaagcg ttcggtgtgt ttgataagga
     6241 tggcaatggc tacatcagtg cagcagagct tcgccacgtg atgacaaacc ttggagagaa
     6301 gttaacagat gaagaggttg atgaaatgat cagggaagca gacatcgatg gggatggtca
     6361 ggtaaactac gaagagtttg tacaaatgat gacagcgaag taagcggccg cactagtata
     6421 acttcgtata atgtatgcta tacgaagtta ttaagaggtt tcatattgct aatagcagct
     6481 acaatccagc taccattctg cataacttcg tataaagtat cctatacgaa gttataagat
     6541 ccaattcttt gccaaaatga tgagacagca caataaccag cacgttgccc aggagctgta
     6601 ggaaaaagaa gaaggcatga acatggttag cagaggctct agagccgccg gtcacacgcc
     6661 agaagccgaa ccccgccctg ccccgtcccc cccgaaggca gccgtccccc cgcggacagc
     6721 cccgagccgc tccccccgca caaagggccc tcccggagcc cctcaaggct ttcacgcagc
     6781 cacagaaaag aaacaagccg tcattaaacc aagcgctaat tacagcccgg aggagaaggg
     6841 ccgtcccgcc cgctcacctg tgggagtaac gcggtcagtc agagccgggg cgggcggcgc
     6901 gaggcggcgg cggagcgggg cacggggcga aggcagcgcg cagcgactcc cgcccgccgc
     6961 gcgcttcgct ttttataggg ccgccgccgc cgccgcctcg ccataaaagg aaactttcgg
     7021 agcgcgccgc tctgattggc tgccgccgca cctctccgcc tcgccccgcc ccgcccctcg
     7081 ccccgccccg ccccgcctgg cgcgcg
//
