LOCUS       sequence-193997-        6414 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bicistronic vector expressing mRuby2 and GCaMP6m from a single open 
            reading frame. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6414)
  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 6414)
  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-GCaMP6m-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..6414
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     32..51
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   368..497
                     /label=AAV2 ITR
     polyA_signal    659..780
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(696..715)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     750..769
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     809..825
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     810..826
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    829..1417
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             935..946
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1344..1364
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(join(1442..1885,1886..1891,1892..2326,2327..2344,
                     2345..2614,2615..2620,2621..2677,2678..2704,2705..2728,
                     2729..2731,2732..2764,2765..2767,2768..2785,2786..2791))
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6m
                     /translation="GSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKT
                     GHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPV
                     LLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEEL
                     FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
                     GVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL
                     KGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSL
                     GQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKGSYRDTEEEIREAFGVFDKDGNG
                     YISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     2001..2020
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2260..2281
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2387..2408)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2689..2698
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2792..2848)
                     /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(2858..3568)
                     /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      3565..3574
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3610..4057)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     repeat_region   4139..4268
                     /label=AAV2 ITR
     rep_origin      4340..4795
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4427..4446)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4637..4658
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4821..4925
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4926..4994,4995..5786)
                     /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(5144..5163)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5957..6414,1..131)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
       61 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      121 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      181 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
      241 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
      301 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
      361 aatgcagctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc
      421 gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc
      481 catcactagg ggttccttgt agttaatgat taacccgcca tgctacttat ctacgtagcc
      541 atgctctagg aagatcctta tcgattttac cacatttgta gaggttttac ttgctttaaa
      601 aaacctccca catctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
      661 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
      721 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
      781 tcatgtctgc tcgaagcggc cgcccgggtc gaggtcgacg gtatcgatgc ggggaggcgg
      841 cccaaaggga gatccgactc gtctgagggc gaaggcgaag acgcggaaga ggccgcagag
      901 ccggcagcag gccgcgggaa ggaaggtccg ctggattgag ggccgaaggg acgtagcaga
      961 aggacgtccc gcgcagaatc caggtggcaa cacaggcgag cagccaagga aaggacgatg
     1021 atttccccga caacaccacg gaattgtcag tgcccaacag ccgagcccct gtccagcagc
     1081 gggcaaggca ggcggcgatg agttccgccg tggcaatagg gagggggaaa gcgaaagtcc
     1141 cggaaaggag ctgacaggtg gtggcaatgc cccaaccagt gggggttgcg tcagcaaaca
     1201 cagtgcacac cacgccacgt tgcctgacaa cgggccacaa ctcctcataa agagacagca
     1261 accaggattt atacaaggag gagaaaatga aagccatacg ggaagcaata gcatgataca
     1321 aaggcattaa agcagcgtat ccacatagcg taaaaggagc aacatagtta agaataccag
     1381 tcaatctttc acaaattttg taatccagag gttgattatc gataagctct agtgcggccg
     1441 cttacttcgc tgtcatcatt tgtacaaact cttcgtagtt tacctgacca tccccatcga
     1501 tgtctgcttc cctgatcatt tcatcaacct cttcatctgt taacttctct ccaaggtttg
     1561 tcatcacgtg gcgaagctct gctgcactga tgtagccatt gccatcctta tcaaacacac
     1621 cgaacgcttc tctaatttct tcttccgtgt ccctgtagct cccttttctt gccatcattg
     1681 tcaggaactc agggaagtcg attgtgccgt caccgtcggc atctacttca ttgatcatgt
     1741 cctgcagctc tgcttctgtg gggttctgcc ccagagaccg catcaccgtc cccagctcct
     1801 tggttgttat tgtcccatcc ccgtccttgt caaataggga gaaagcctct ttaaattctg
     1861 cgatctgctc ttcagtcagt tggtccggca ggttgtactc cagcttgtgc cccaggatgt
     1921 tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg gttcaccagg gtgtcgccct
     1981 cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct
     2041 cctggatgta gccttcgggc atggcggact tgaagaagtc gtgctgcttc atgtggtcgg
     2101 ggtagcggct gaagcactgc acgccgtagg tcagggtggt cacgagggtg ggccagggca
     2161 cgggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgtag gtggcatcgc
     2221 cctcaccctc gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca
     2281 ggatgggcac caccccggtg aacagctcct cgcccttgct caccatgctc cctccggtac
     2341 cgcccttgta cagctcgtcc atgccgagag tgatcccggc ggcggtcacg aactccagca
     2401 ggaccatgtg atcgcgcttc tcgttggggt ctttcgaaag tttggactgc acgctcaggt
     2461 agtggttgtc gggcagcagc acggggccgt cgccgatggg ggtgttctgc tggtagtggt
     2521 aggcgagctg cacgccgccg tcctcgatgt tgtggcggat cttgaagttc gccttgatgc
     2581 cgttcttctg cttgtcggcc ttgatataga cgttctcgag tgagctcagc cgacctatag
     2641 ctctgactgc gtgacctgtc ttattccact tacgacgtga tgagtcgacc atggtggcga
     2701 gatccttatc gtcatcgtcg tacagatccc gacccatttg ctgtccacca gtcatgctag
     2761 ccataccatg atgatgatga tgatgagaac caggaccggg gttttcttcc acgtctcctg
     2821 cttgctttaa cagagagaag ttcgtggctc cggatccctt gtacagctcg tccatcccac
     2881 caccaagccc ggcgaacttg gcaactgcgt gttcgcgttg tactacgaac atttcattgt
     2941 cactttcctc taacctttcc aggcggtgat caacggcatg gataccgggc atcttgatgt
     3001 tcccgacggt cttttttgac ctgtaagttg ttacgaaaga gcaagacaga tggccaccac
     3061 catcaacttt cagtgccata tgagtgtatc ccctcagacc accatctgct ggatacatca
     3121 tctctgtatt aggctcccaa cccttggtct tcttctgcat cacgggacca ttggagggaa
     3181 agtttacccc tctgacttgg acgtggtaaa cgagacagcc atcctcaagg ctggtgtcct
     3241 gcatgacggt gacgactcca ccatcttcgt atctcgtaac tctttcccaa gtaaaaccct
     3301 caggaaagga ctgtttaaag aaatcaggaa tgcctttcgg gtacttgata aaagtacggc
     3361 tgccatacat gaacgacgtg gcaagaatgt caaaggcaaa tggcaggggt cctccctcga
     3421 tgactttgat cctcatggtt tgagttccca tgtacggatt gccttctcct tcacctgtgc
     3481 atttgaattg gtggccgttg accgaacctt ccatgaccac cttcatacgc atattttcct
     3541 tgatcagctc ttcgccctta gacaccatgg tggccctagg accggtacta gctagcagct
     3601 tgaattcgac tgcgctctca ggcacgacac gactcctccg ctgcccaccg cagactgagg
     3661 cagcgctgag tcgccggcgc cgcagcgcag atggtcgcgc ccgtgccccc ctatctcgcg
     3721 cctcgcgtgg tgcggtccgg ctgggccggc ggcggcgcgg acgcgaccaa ggtggccggg
     3781 aaggggagtt tgcgggggac cggcgagtga cgtcagcgcg ccttcagtgc tgaggcggcg
     3841 gtggcgcgcg ccgccaggcg ggggcgaagg cactgtccgc ggtgctgaag ctggcagtgc
     3901 gcacgcgcct cgccgcatcc tgtttcccct ccccctctct gataggggat gcgcaatttg
     3961 gggaatgggg gttgggtgct tgtccagtgg gtcggggtcg gtcgtcaggt aggcaccccc
     4021 accccgcctc atcctggtcc taaaacccac ttgcactcat acgcagggcc ctctgcagag
     4081 atcttcctag agcatggcta cgtagataag tagcatggcg ggttaatcat taactacaag
     4141 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     4201 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     4261 gcgcgcagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     4321 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4381 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     4441 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     4501 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     4561 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     4621 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     4681 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     4741 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     4801 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4861 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4921 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4981 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5041 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5101 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5161 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5221 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5281 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     5341 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     5401 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     5461 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     5521 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     5581 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     5641 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     5701 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     5761 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     5821 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     5881 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     5941 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6001 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6061 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6121 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6181 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6241 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6301 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     6361 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcg
//
