LOCUS       sequence-198992-        7080 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Cre-dependent bicistronic vector expressing mRuby2 and GCaMP6f from 
            a single open reading frame..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7080)
  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 7080)
  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-GCaMP6f-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..7080
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     411..433
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     495..514
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(548..581)
                     /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(632..665)
                     /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(680..1123,1124..1129,1130..1564,1565..1582,
                     1583..1852,1853..1858,1859..1915,1916..1942,1943..1966,
                     1967..1969,1970..2002,2003..2005,2006..2023,2024..2029))
                     /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"
     primer_bind     1239..1258
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1498..1519
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1625..1646)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      1927..1936
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2030..2086)
                     /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(2096..2806)
                     /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      2803..2812
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2837..2870
                     /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    2921..2954
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2967..3555
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3020..3040)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3438..3449)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3598..3719
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3609..3628)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3663..3682
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3881..4010
                     /label=AAV2 ITR
     rep_origin      complement(4247..4835)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4327..4346)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5006..5797,5798..5866))
                     /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     5629..5648
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5867..5971)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5997..6452)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6134..6155)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6346..6365
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6524..6653
                     /label=AAV2 ITR
ORIGIN
        1 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg
       61 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
      121 gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc
      181 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac
      241 ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg
      301 tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg
      361 gggctgtccg cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt
      421 ctggcgtgtg accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt
      481 cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt
      541 ggatcttata acttcgtata ggatacttta tacgaagtta tgcagaatgg tagctggatt
      601 gtagctgcta ttagcaatat gaaacctctt aataacttcg tatagcatac attatacgaa
      661 gttatactag tgcggccgct tacttcgctg tcatcatttg tacaaactct tcgtagttta
      721 cctgaccatc cccatcgatg tctgcttccc tgatcatttc atcaacctct tcatctgtta
      781 acttctctcc aaggtttgtc atcacgtggc gaagctctgc tgcactgatg tagccattgc
      841 catccttatc aaacacaccg aacgcttctc taatttcttc ttccgtgtcc ctgtatttca
      901 tttttcttgc catcattgtc aggaactcag ggaagtcgat tgtgccgtca ccgtcggcat
      961 ctacttcatt gatcatgtcc tgcagctctg cttctgtggg gttctgcccc agagaccgca
     1021 tcaccgtccc cagctccttg gttgttattg tcccatcccc gtccttgtca aatagggaga
     1081 attcctcttt aaattctgcg atctgctctt cagtcagttg gtccggcagg ttgtactcca
     1141 gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt
     1201 tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct
     1261 tgaagaagat ggtgcgctcc tggatgtagc cttcgggcat ggcggacttg aagaagtcgt
     1321 gctgcttcat gtggtcgggg tagcggctga agcactgcac gccgtaggtc agggtggtca
     1381 cgagggtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct
     1441 tgccgtaggt ggcatcgccc tcaccctcgc cggacacgct gaacttgtgg ccgtttacgt
     1501 cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca
     1561 ccatgctccc tccggtaccg cccttgtaca gctcgtccat gccgagagtg atcccggcgg
     1621 cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct ttcgaaagtt
     1681 tggactgcac gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg
     1741 tgttctgctg gtagtggtag gcgagctgca cgccgccgtc ctcgatgttg tggcggatct
     1801 tgaagttcgc cttgatgccg ttcttctgct tgtcggcctt gatatagacg ttctcgagtg
     1861 agctcagccg acctatagct ctgactgcgt gacctgtctt attccactta cgacgtgatg
     1921 agtcgaccat ggtggcgaga tccttatcgt catcgtcgta cagatcccga cccatttgct
     1981 gtccaccagt catgctagcc ataccatgat gatgatgatg atgagaacca ggaccggggt
     2041 tttcttccac gtctcctgct tgctttaaca gagagaagtt cgtggctccg gatcccttgt
     2101 acagctcgtc catcccacca ccaagcccgg cgaacttggc aactgcgtgt tcgcgttgta
     2161 ctacgaacat ttcattgtca ctttcctcta acctttccag gcggtgatca acggcatgga
     2221 taccgggcat cttgatgttc ccgacggtct tttttgacct gtaagttgtt acgaaagagc
     2281 aagacagatg gccaccacca tcaactttca gtgccatatg agtgtatccc ctcagaccac
     2341 catctgctgg atacatcatc tctgtattag gctcccaacc cttggtcttc ttctgcatca
     2401 cgggaccatt ggagggaaag tttacccctc tgacttggac gtggtaaacg agacagccat
     2461 cctcaaggct ggtgtcctgc atgacggtga cgactccacc atcttcgtat ctcgtaactc
     2521 tttcccaagt aaaaccctca ggaaaggact gtttaaagaa atcaggaatg cctttcgggt
     2581 acttgataaa agtacggctg ccatacatga acgacgtggc aagaatgtca aaggcaaatg
     2641 gcaggggtcc tccctcgatg actttgatcc tcatggtttg agttcccatg tacggattgc
     2701 cttctccttc acctgtgcat ttgaattggt ggccgttgac cgaaccttcc atgaccacct
     2761 tcatacgcat attttccttg atcagctctt cgcccttaga caccatggtg gccctaggac
     2821 cggtactagt ctcgagataa cttcgtataa agtatcctat acgaagttat ttgccttaac
     2881 ccagaaatta tcactgttat tctttagaat ggtgcaaaga ataacttcgt ataatgtatg
     2941 ctatacgaag ttataagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat
     3001 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     3061 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     3121 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     3181 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     3241 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     3301 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     3361 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     3421 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     3481 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     3541 gggccgcctc cccgcatcga taccgtcgac ccgggcggcc gcttcgagca gacatgataa
     3601 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     3661 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     3721 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt
     3781 aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatcttcct agagcatggc
     3841 tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag
     3901 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     3961 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcattaat
     4021 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4081 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4141 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4201 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4261 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4321 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4381 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4441 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4501 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4561 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4621 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4681 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4741 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4801 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4861 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4921 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4981 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5041 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5101 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5161 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5221 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5281 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5341 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5401 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5461 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5521 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5581 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5641 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5701 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5761 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5821 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5881 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     5941 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat
     6001 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     6061 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     6121 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     6181 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     6241 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     6301 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     6361 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     6421 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg
     6481 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctgcgcg ctcgctcgct
     6541 cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt
     6601 gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt ccttgtagtt
     6661 aatgattaac ccgccatgct acttatctac gtagccatgc tctaggaaga gtaccattga
     6721 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     6781 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     6841 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     6901 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     6961 tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc
     7021 caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg
//
