LOCUS       sequence-474735-        7131 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated and Cre-dependent expression of jGCaMP8m and CyRFP1 in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7131)
  TITLE     Dendritic compartment specific spine formation in layer 5 neurons 
            underlies cortical circuit maturation during adolescence
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7131)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-CAG-FLEX-jGCaMP8m-P2A-CyRFP1-WPRE
FEATURES             Location/Qualifiers
     source          1..7131
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   2..131
                     /label=AAV2 ITR
     promoter        442..719
                     /label=chicken beta-actin promoter
     primer_bind     998..1020
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1082..1101
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     1139..1158
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     protein_bind    1169..1202
                     /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    1265..1298
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1321..2019)
                     /codon_start=1
                     /product="cyan-excitable red fluorescent protein (Laviv et 
                     al., 2016)"
                     /label=CyRFP1
                     /translation="VSKGEELIKENMRSKLYLEGSVNGHQFKCTHEGEGKPYEGKQTNR
                     IKVVEGGPLPFAFDILATMFMYGSKVFIKYPADLPDYFKQSFPEGFTWERVMVFEDGGV
                     LTATQDTSLQDGELIYNVKLRGVNFPANGPVMQKKTLGWEPSTETMYPADGGLEGRCDK
                     ALKLVGGGHLHVNFKTTYKSKKPVKMPGVHYVDRRLERIKEADNETYVEQYEHAVARYS
                     NLGGGMDELYK"
     CDS             complement(2020..2076)
                     /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"
     primer_bind     2642..2661
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2901..2922
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3028..3049)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(3331..3348)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(3386..3419)
                     /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(3490..3523)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3576..4164
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3629..3649)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4047..4058)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4207..4328
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4218..4237)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4272..4291
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4490..4619
                     /label=AAV2 ITR
     rep_origin      complement(4856..5444)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4936..4955)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5615..6406,6407..6475))
                     /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     6238..6257
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6476..6580)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6606..7061)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6743..6764)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6955..6974
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt
       61 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      121 taggggttcc ttgtagttaa tgattaaccc gccatgctac ttatctacgt agccatgctc
      181 taggaagagt accattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      241 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      301 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      361 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      421 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      481 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      541 tgggggcggg gggggggggg gggcgcgcgc cagggggggg gggggggggg gagggggggg
      601 gsggggsgag gsggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc
      661 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg
      721 gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
      781 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
      841 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa
      901 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ctgtccgcgg
      961 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1021 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1081 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgga tccggtactt
     1141 gcctttctct ccacaggtga tgatccccat aacttcgtat aaagtatcct atacgaagtt
     1201 atatcaaaat aggaagacca atgcttcacc atcgacccga attgccaagc atcaccatcg
     1261 acacataact tcgtataatg tatgctatac gaagttatac taggagctcg gcgcgcctca
     1321 cttgtacagc tcgtccatgc ctccgcccag gttggagtat ctggccacag cgtgctcgta
     1381 ctgctcgacg taggtctcgt tgtcggcctc cttgattctt tccagtctgc ggtccacgta
     1441 gtggacgccg ggcatcttca cgggtttctt ggacttgtat gtggtcttga agttgacgtg
     1501 caggtggccc ccgcccacga gcttcagggc cttgtcgcat ctgccttcca ggccgccgtc
     1561 agcggggtac atggtctcgg tgctgggctc ccagcccagt gttttcttct gcatcacggg
     1621 gccgttggct gggaagttca cccctctgag cttgacgttg tagatgagct cgccgtcctg
     1681 gaggctggtg tcctgggtgg cggtcagcac gcccccgtct tcgaacacca tgactctctc
     1741 ccatgtgaag ccctcaggga aggactgctt aaaataatcg gggaggtcgg cggggtactt
     1801 gatgaacacc ttgctcccgt acataaacat ggtggccagg atgtcgaatg cgaacggcag
     1861 ggggcctccc tccaccacct tgatcctgtt ggtctgcttg ccctcgtagg gcttgccctc
     1921 cccttcgtgg gtgcacttga actggtggcc gttcacgctg ccttccaggt acagcttgct
     1981 tctcatgttc tccttgatca gctcctcgcc cttgctcaca ggaccggggt tttcttccac
     2041 gtctcctgct tgctttaaca gagagaagtt cgtggctccg gatcccttcg ctgtcatcat
     2101 ttgtacaaac tcttcgtagt ttacctgacc atccccatcg atgtctgctt ccctgatcat
     2161 ttcatcaacc tcttcatctg ttaacttctc tccaaggttt gtcatcacgt ggcgaagctc
     2221 tgctgcactg atgtagccat tgccatcctt atcaaacaca ccgaacgctt ctctaatttc
     2281 ttcttccgtg tccctgtatt tcatttttct tgccatcatt gtcaggaact cagggaagtc
     2341 gattgtgccg tcaccgtcgg catctacttc attgatcatg tcctgcagct ctgcttctgt
     2401 ggggttctgc cccagagacc gcatcaccgt ccccagctcc ttggttgtta ttgtcccatc
     2461 cccgtccttg tcaaataggg agaaagcctc tttgtattct gcgatctgct cttcagtcag
     2521 ttggtccggc aggttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc
     2581 gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg
     2641 ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc tcctggatgt agccttcggg
     2701 catggcggac ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc tgaagcactg
     2761 cacgccgtag gtcagggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt
     2821 gcagatgaac ttcagggtca gcttgccgta ggtggcatcg ccctcaccct cgccggacac
     2881 gctgaacttg tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt
     2941 gaacagctcc tcgcccttgc tcaccatgct ccctccggta ccgcccttgt acagctcgtc
     3001 catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt
     3061 ctcgttgggg tctttcgaaa gtttggactc cacgctcagg tagtggttgt cgggcagcag
     3121 cacggggccg tcgccgatgg gggtgttctg ctggtagtgg taggcgagct gcacgccgcc
     3181 gtcctcgatg ttgtggcgga tgtggaagtt cgccttgatg ccgttcttct gcttgtcggc
     3241 cttgatatag acgttgatct tcaggcccat caggcggccg atgatcttca cggcggtggc
     3301 cacctccttg aaggtcttct tgcgacgcgt gtgatgatgg tgatgatgca tggtggcact
     3361 agtgctagcg gtaccgaatt ctagtataac ttcgtatagg atactttata cgaagttatc
     3421 attgggattc ttcctatttt gatccaagca tcaccatcga ccctctagtc cagatctcac
     3481 catcgaccca taacttcgta tagcatacat tatacgaagt tatgtccctc gaagaggttc
     3541 gaattcgata tcaagcttat cgatagctta tcgataatca acctctggat tacaaaattt
     3601 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     3661 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     3721 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     3781 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     3841 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     3901 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     3961 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc
     4021 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     4081 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     4141 tctccctttg ggccgcctcc ccgcatcgat accgtcgacc cgggcggccg cttcgagcag
     4201 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     4261 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     4321 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg
     4381 aggtttttta aagcaagtaa aacctctaca aatgtggtaa aatcgataag gatcttccta
     4441 gagcatggct acgtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta
     4501 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     4561 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     4621 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4681 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4741 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4801 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4861 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4921 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4981 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5041 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5101 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5161 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5221 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5281 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5341 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5401 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5461 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5521 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5581 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5641 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5701 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5761 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5821 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5881 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5941 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6001 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6061 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6121 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6181 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6241 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6301 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6361 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6421 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6481 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6541 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6601 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     6661 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     6721 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     6781 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     6841 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     6901 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     6961 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     7021 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
     7081 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc a
//
