LOCUS       sequence-76642-e        6756 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 6756)
  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 6756)
  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..6756
                     /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      520..529
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             526..1236
                     /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             1246..1302
                     /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(1303..1308,1309..1326,1327..1329,1330..1362,1363..1365,
                     1366..1389,1390..1416,1417..1473,1474..1479,1480..1749,
                     1750..1767,1768..2202,2203..2208,2209..2652)
                     /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      1396..1405
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1686..1707
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1813..1834)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2074..2093)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2696..3284
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2749..2769)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3167..3178)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3287..3303)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3288..3304)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3333..3454
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3344..3363)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3398..3417
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3616..3745
                     /label=AAV2 ITR
     primer_bind     complement(3764..3781)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3764..3780)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3773..3795)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3922..4377
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4009..4028)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4219..4240
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4403..4507
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4508..4576,4577..5368)
                     /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(4726..4745)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5539..6127
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6028..6047
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6415..6436
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6451..6456,6457..6474,6475..6481)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6489..6505
                     /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     6494..6516
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6513..6529
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6513..6529
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   6567..6696
                     /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 ctgctagcaa ggatccaccc gccacctacc ggtcctaggg ccaccatggt gtctaagggc
      541 gaagagctga tcaaggaaaa tatgcgtatg aaggtggtca tggaaggttc ggtcaacggc
      601 caccaattca aatgcacagg tgaaggagaa ggcaatccgt acatgggaac tcaaaccatg
      661 aggatcaaag tcatcgaggg aggacccctg ccatttgcct ttgacattct tgccacgtcg
      721 ttcatgtatg gcagccgtac ttttatcaag tacccgaaag gcattcctga tttctttaaa
      781 cagtcctttc ctgagggttt tacttgggaa agagttacga gatacgaaga tggtggagtc
      841 gtcaccgtca tgcaggacac cagccttgag gatggctgtc tcgtttacca cgtccaagtc
      901 agaggggtaa actttccctc caatggtccc gtgatgcaga agaagaccaa gggttgggag
      961 cctaatacag agatgatgta tccagcagat ggtggtctga ggggatacac tcatatggca
     1021 ctgaaagttg atggtggtgg ccatctgtct tgctctttcg taacaactta caggtcaaaa
     1081 aagaccgtcg ggaacatcaa gatgcccggt atccatgccg ttgatcaccg cctggaaagg
     1141 ttagaggaaa gtgacaatga aatgttcgta gtacaacgcg aacacgcagt tgccaagttc
     1201 gccgggcttg gtggtgggat ggacgagctg tacaagggat ccggagccac gaacttctct
     1261 ctgttaaagc aagcaggaga cgtggaagaa aaccccggtc ctggttctca tcatcatcat
     1321 catcatggta tggctagcat gactggtgga cagcaaatgg gtcgggatct gtacgacgat
     1381 gacgataagg atctcgccac catggtcgac tcatcacgtc gtaagtggaa taagacaggt
     1441 cacgcagtca gagctatagg tcggctgagc tcactcgaga acgtctatat caaggccgac
     1501 aagcagaaga acggcatcaa ggcgaacttc aagatccgcc acaacatcga ggacggcggc
     1561 gtgcagctcg cctaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1621 cccgacaacc actacctgag cgtgcagtcc aaactttcga aagaccccaa cgagaagcgc
     1681 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1741 ctgtacaagg gcggtaccgg agggagcatg gtgagcaagg gcgaggagct gttcaccggg
     1801 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1861 ggcgagggtg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1921 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     1981 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     2041 ggctacatcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     2101 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     2161 aaggaggacg gcaacatcct ggggcacaag ctggagtaca acctgccgga ccaactgact
     2221 gaagagcaga tcgcagaatt taaagaggaa ttctccctat ttgacaagga cggggatggg
     2281 acaataacaa ccaaggagct ggggacggtg atgcggtctc tggggcagaa ccccacagaa
     2341 gcagagctgc aggacatgat caatgaagta gatgccgacg gtgacggcac aatcgacttc
     2401 cctgagttcc tgacaatgat ggcaagaaaa atgaaataca gggacacgga agaagaaatt
     2461 agagaagcgt tcggtgtgtt tgataaggat ggcaatggct acatcagtgc agcagagctt
     2521 cgccacgtga tgacaaacct tggagagaag ttaacagatg aagaggttga tgaaatgatc
     2581 agggaagcag acatcgatgg ggatggtcag gtaaactacg aagagtttgt acaaatgatg
     2641 acagcgaagt aagcggccgc actagctagt gcggccgctt atgaaagcta tcgataatca
     2701 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     2761 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     2821 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     2881 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     2941 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     3001 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     3061 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg
     3121 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     3181 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     3241 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgtcgacc
     3301 tcgacccggg cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac
     3361 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     3421 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     3481 gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     3541 tggtaaaatc gataaggatc ttcctagagc atggctacgt agataagtag catggcgggt
     3601 taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     3661 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     3721 cctcagtgag cgagcgagcg cgcagcctta attaacctaa ttcactggcc gtcgttttac
     3781 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     3841 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     3901 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3961 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     4021 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4081 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     4141 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     4201 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     4261 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     4321 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     4381 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4441 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4501 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     4561 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     4621 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     4681 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4741 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4801 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4861 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4921 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4981 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5041 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     5101 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     5161 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     5221 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5281 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5341 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     5401 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5461 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5521 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5581 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5641 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5701 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5761 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5821 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5881 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5941 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6001 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6061 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6121 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     6181 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     6241 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     6301 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     6361 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     6421 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     6481 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
     6541 cagatttaat taaggcctta attaggctgc gcgctcgctc gctcactgag gccgcccggg
     6601 caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca
     6661 gagagggagt ggccaactcc atcactaggg gttccttgta gttaatgatt aacccgccat
     6721 gctacttatc tacgtagcca tgctctagga agatct
//
