LOCUS       sequence-343479-        7113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent GCaMP6s and mBeRFP expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7113)
  AUTHORS   Melzer S, Newmark ER, Mizuno GO, Hyun M, Philson AC, Quiroli E, 
            Righetti B, Gregory MR, Huang KW, Levasseur J, Tian L, Sabatini BL
  TITLE     Bombesin-like peptide recruits disinhibitory cortical circuits and 
            enhances fear memories.
  JOURNAL   Cell. 2021 Oct 28;184(22):5622-5634.e25. doi: 
            10.1016/j.cell.2021.09.013. Epub 2021 Oct 4.
  PUBMED    34610277
REFERENCE   2  (bases 1 to 7113)
  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: 
            "10.1016/j.cell.2021.09.013"; journalName: "Cell"; date: 
            "2021-10-28- 28"; volume: "184"; issue: "22"; pages: "5622-5634.e25"
COMMENT     Sequence Label: pAAV-CBA-DIO-GCaMP6s-P2A-mBeRFP-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..7113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     807..829
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     891..910
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(944..977)
                     /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(1028..1061)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1083..1796)
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERSTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPADGGLEGRDY
                     MALKLVGGGHLICNAKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETSVEQHEVAV
                     ARYCDLPSKLGHKL*"
     CDS             complement(1083..1796)
                     /codon_start=1
                     /product="monomeric photoactivatable red fluorescent 
                     protein (Gunewardene et al., 2011)"
                     /label=PAmKate
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERSTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPADGGLEGRDY
                     MALKLVGGGHLICNAKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETSVEQHEVAV
                     ARYCDLPSKLGHKL*"
     CDS             complement(1797..1853)
                     /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(join(1863..2303,2304..2309,2310..2744,2745..2762,
                     2763..3032,3033..3038,3039..3095,3096..3122,3123..3146,
                     3147..3149,3150..3182,3183..3185,3186..3203,3204..3212))
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     2419..2438
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2678..2699
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2805..2826)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      3107..3116
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3209..3218
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3237..3270
                     /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    3321..3354
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3367..3955
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3420..3440)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3838..3849)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3998..4119
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4009..4028)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4063..4082
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4281..4410
                     /label=AAV2 ITR
     rep_origin      complement(4647..5235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4727..4746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5406..6197,6198..6266))
                     /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     6029..6048
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6267..6371)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6397..6852)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6534..6555)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6746..6765
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6924..7053
                     /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 rggggggggg gggggggggg gggggggggg sggggsgrgg
      421 sggrgaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
      481 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
      541 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga
      601 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
      661 tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg
      721 ctccgggagg gccctttgtg cggggggagc ggctcggggc tgtccgcggg gggacggctg
      781 ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag
      841 agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct
      901 ggttattgtg ctgtctcatc attttggcaa agaattggat cttataactt cgtataggat
      961 actttatacg aagttatgca gaatggtagc tggattgtag ctgctattag caatatgaaa
     1021 cctcttaata acttcgtata gcatacatta tacgaagtta tactagtgcg gccgcggaat
     1081 tcttattaaa gtttgtgccc cagtttgcta gggaggtcgc agtatctggc cacagccacc
     1141 tcgtgctgct cgacggaggt ctctttgtcg gcctccttga ttctttccag tcttctgtcc
     1201 acatagtaga cgccgggcat cttgaggttc ttagcgggtt tcttggatct gtatgtggtc
     1261 ttggcgttgc agatcaggtg gcccccgccc acgagcttca gggccatgta gtctctgcct
     1321 tccaggccgc cgtcagcggg gtacagcatc tcggtgctgg cctcccagcc gagtgttttc
     1381 ttctgcatca cagggccgtt ggatgggaag ttcacccctc tgatcttgac gttgtagatg
     1441 aggcagccgt cctggaggct ggtgtcctgg gtagcggtca gcacgccccc gtcttcgtat
     1501 gtggtggatc tctcccatgt gaagccctca gggaaggact gcttaaagaa gtcggggatg
     1561 ccctgggtgt ggttgatgaa ggtcttgctg ccgtacatga agctggtagc caggatgtcg
     1621 aaggcgaagg ggagagggcc gccctcgacc accttgattc tcatggtctg ggtgccctcg
     1681 tagggcttgc cttcgccctc ggatgtgcac ttgaagtggt ggttgttcac ggtgccctcc
     1741 atgtacagct tcatgtgcat gttctcctta atcagctctt cgcccttaga caccatagga
     1801 ccggggtttt cttccacgtc tcctgcttgc tttaacagag agaagttcgt ggctccggat
     1861 cccttcgctg tcatcatttg tacaaactct tcgtagttta cctgaccatc cccatcgatg
     1921 tctgcttccc tgatcatttc atcaacctct tcatctgtta acttctctcc aaggtttgtc
     1981 atcacgtggc gaagctctgc tgcactgatg tagccattgc catccttatc aaacacaccg
     2041 aacgcttctc taatttcttc ttccgtgtcc ctgtatttca tttttcttgc catcattgtc
     2101 aggaactcag ggaagtcgat tgtgccgtca ccgtcggcat ctacttcatt gatcatgtcc
     2161 tgcagctctg cttctgtggg gttctgcccc agagaccgca tcaccgtccc cagctccttg
     2221 gttgttattg tcccatcccc gtccttgtca aatagggaga aagcctcttt aaattctgcg
     2281 atctgctctt cagtcagttg gtccggcagg ttgtactcca gcttgtgccc caggatgttg
     2341 ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg
     2401 aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc
     2461 tggatgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg
     2521 tagcggctga agcactgcac gccgtaggtc agggtggtca cgagggtggg ccagggcacg
     2581 ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt ggcatcgccc
     2641 tcaccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg
     2701 atgggcacca ccccggtgaa cagctcctcg cccttgctca ccatgctccc tccggtaccg
     2761 cccttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg
     2821 accatgtgat cgcgcttctc gttggggtct ttcgaaagtt tggactgcac gctcaggtag
     2881 tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtag
     2941 gcgagctgca cgccgccgtc ctcgatgttg tggcggatgt ggaagttcgc cttgatgccg
     3001 ttcttctgct tgtcggcctt gatatagacg ttctcgagtg agctcagccg acctatagct
     3061 ctgactgcgt gacctgtctt attccactta cgacgtgatg agtcgaccat ggtggcgaga
     3121 tccttatcgt catcgtcgta cagatcccga cccatttgct gtccaccagt catgctagcc
     3181 ataccatgat gatgatgatg atgagaaccc atggtggcac cggtactagt ctcgagataa
     3241 cttcgtataa agtatcctat acgaagttat ttgccttaac ccagaaatta tcactgttat
     3301 tctttagaat ggtgcaaaga ataacttcgt ataatgtatg ctatacgaag ttataagctt
     3361 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     3421 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     3481 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     3541 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     3601 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     3661 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     3721 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     3781 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     3841 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     3901 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     3961 taccgtcgac ccgggcggcc gcttcgagca gacatgataa gatacattga tgagtttgga
     4021 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     4081 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     4141 tttatgtttc aggttcaggg ggagatgtgg gaggtttttt aaagcaagta aaacctctac
     4201 aaatgtggta aaatcgataa ggatcttcct agagcatggc tacgtagata agtagcatgg
     4261 cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg
     4321 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     4381 ggcggcctca gtgagcgagc gagcgcgcag ctgcattaat gaatcggcca acgcgcgggg
     4441 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4501 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4561 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4621 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4681 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4741 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4801 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4861 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4921 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4981 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5041 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5101 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5161 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5221 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5281 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5341 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5401 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5461 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5521 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5581 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5641 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5701 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5761 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5821 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5881 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5941 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6001 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6061 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6121 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6181 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6241 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6301 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6361 ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt
     6421 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     6481 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     6541 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     6601 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     6661 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     6721 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     6781 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     6841 tacagggcgc gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     6901 gggcctcttc gctattacgc cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa
     6961 agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag
     7021 agggagtggc caactccatc actaggggtt ccttgtagtt aatgattaac ccgccatgct
     7081 acttatctac gtagccatgc tctaggaaga gta
//
