LOCUS       sequence-463042-        6055 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AAV vector expressing the mScarlet based calcium indicator 
            PinkyCaMP under Syn promoter in a Cre-dependent manner (DIO, double 
            floxed inverted open reading frame).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6055)
  AUTHORS   Fink R, Imai S, Gockel N, Lauer G, Renken K, Wietek J, 
            Lamothe-Molina PJ, Furhmann F, Mittag M, Ziebarth T, Canziani A, 
            Kubitschke M, Kistmacher V, Kretschmer A, Sebastian E, Schmitz D, 
            Terai T, Grundemann J, Hassan S, Patriarchi T, Reiner A, Fuhrmann M,
            Campbell RE, Masseck OA
  TITLE     PinkyCaMP a mScarlet-based calcium sensor with exceptional 
            brightness, photostability, and multiplexing capabilities.
  JOURNAL   bioRxiv [Preprint]. 2025 Jan 11:2024.12.16.628673. doi: 
            10.1101/2024.12.16.628673.
  PUBMED    39763884
REFERENCE   2  (bases 1 to 6055)
  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: "bioRxiv 
            [Preprint]. 2025 Jan 11:2024.12.16.628673. doi: 
            10.1101/2024.12.16.628673."
COMMENT     Sequence Label: pAAV-Syn-DIO-PinkyCaMP
FEATURES             Location/Qualifiers
     source          1..6055
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     142..161
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   320..449
                     /label=AAV2 ITR
     promoter        549..996
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    1009..1042
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1113..1146
                     /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)."
     CDS             complement(1147..1590)
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTFDFPEFLTMMARKMNDTDSEEEIREAFRVFDKDGNGYIGAAEL
                     RHVMTDLGEKLTDEEVDEMIRVADIDGDGQVNYEEFVQMMTAK"
     primer_bind     complement(1620..1639)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             complement(2326..2382)
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     CDS             complement(join(2425..2439,2440..2448))
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             complement(2452..2484)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             complement(2488..2505)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(2572..2605)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(2668..2701)
                     /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)."
     misc_feature    2726..3314
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2779..2799)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3197..3208)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3355..3562
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3732..3861
                     /label=AAV2 ITR
     rep_origin      complement(4098..4686)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4178..4197)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4857..5648,5649..5717))
                     /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     5480..5499
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5718..5822)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5848..6055,1..248))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5985..6006)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
       61 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
      121 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
      181 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
      241 gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
      301 ctcttcgcta ttacgccagc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc
      361 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
      421 agtggccaac tccatcacta ggggttcctt gtagttaatg attaacccgc catgctactt
      481 atctacgtag ccatgctcta ggaagatcgt aggttaatta atctagactg cagagggccc
      541 tgcgtatgag tgcaagtggg ttttaggacc aggatgaggc ggggtggggg tgcctacctg
      601 acgaccgacc ccgacccact ggacaagcac ccaaccccca ttccccaaat tgcgcatccc
      661 ctatcagaga gggggagggg aaacaggatg cggcgaggcg cgtgcgcact gccagcttca
      721 gcaccgcgga cagtgccttc gcccccgcct ggcggcgcgc gccaccgccg cctcagcact
      781 gaaggcgcgc tgacgtcact cgccggtccc ccgcaaactc cccttcccgg ccaccttggt
      841 cgcgtccgcg ccgccgccgg cccagccgga ccgcaccacg cgaggcgcga gatagggggg
      901 cacgggcgcg accatctgcg ctgcggcgcc ggcgactcag cgctgcctca gtctgcggtg
      961 ggcagcggag gagtcgtgtc gtgcctgaga gcgcagtcga gaggatccat aacttcgtat
     1021 aatgtatgct atacgaagtt atgggtcgat ggtgagatct ggactagagg gtcgatggtg
     1081 atgcttggat caaaatagga agaatcccaa tgataacttc gtataaagta tcctatacga
     1141 agttattcac ttcgctgtca tcatttgtac aaactcttcg tagtttacct gaccatcccc
     1201 atcgatgtct gctaccctga tcatttcatc aacctcctca tctgttaact tctctccaag
     1261 gtctgtcatc acgtggcgaa gctctgctgc gccgatgtag ccattgccgt ccttatcaaa
     1321 cacgcggaac gcttctctaa tttcctcttc actgtctgtg tcattcattt ttcttgccat
     1381 catcgtcagg aactcaggga agtcgaatgt gccgtcaccg tcggcatcta cttcattgat
     1441 catgtcctgc agctctgctt ctgtggggtt ctgccccaga gaccgcatca ccgtccccag
     1501 ctccttggtt gttatcgtcc catccccgtc cttgtcaaat agggagaaag cctctttaaa
     1561 ttctgcgatc tgctcttcag tcagttggtc cagaataaac gccggcatcc agcccattgt
     1621 cttcttctgc attacggggc cgtcgggggg gaagttggtg ccgcgcagct tcaccttgta
     1681 gatgaatacg ccgtcctcca gggaggtgtc ctgaaacaca accacggcgc cgccgtcctc
     1741 gaagttcatc acgcgctccc acttgaagcc ctcggggaag gactgcttat agtagtcggg
     1801 gatgtcggcg gggtgcttag tgaaggccct ggagccgtac atgaactgag gggacaggat
     1861 gtcccaggag aagggcaggg ggccaccctt ggtcaccgtc agcttggcgg tctgggtacc
     1921 ctcgtagggg cggccctcgc cctcgccctc gatctcgaac tcgtggccgt ttacggagcc
     1981 ctccatgtgc accttgtagc gcatgaactc cttgatcact gcctcgccct tgctcaccat
     2041 actaccacca gttcctccct tgtacagctc gtccatgccg ccggtggagt ggcgcgccac
     2101 ggagcgttcg tactgttcca ccacggtgta gtcctcgttg tgggaggtga tgtccaactt
     2161 gcggtcgacg ttgtaggcgc cgggcatctg cacgggcttc ttggcaatat aggtggtctt
     2221 gaagtccgcc aggtagcggc cgccgtcctt caggcgcagg gccatcttaa tgtcgccctt
     2281 cagcacgccg tcctcggggt acaaccgctc cacagaccaa gatcttgagc tcagccgacc
     2341 tatagctctg actgcgtgac ctgccttatt ccacttacga cgtgatgagt cgaccattga
     2401 tgagtcgacc attgttgcga gatccttatc gtcatcgtcg tacagatccc gacccatttg
     2461 ctgtccacca gtcatgctag ccataccatg atgatgatga tgatgagaac ctcctccagt
     2521 cttgttaata tcaagtccag ccaacttaag agcaagctcg ttctgcagca tataacttcg
     2581 tatagcatac attatacgaa gttatgggtc gatggtgatg cttggcaatt cgggtcgatg
     2641 gtgaagcatt ggtcttccta ttttgatata acttcgtata ggatacttta tacgaagtta
     2701 tgaattcgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     2761 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     2821 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     2881 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     2941 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     3001 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     3061 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     3121 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     3181 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     3241 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     3301 ggccgcctcc ccgcatcgat accgtcgacc cgggcggccg ctcataaagc tcgactgtgc
     3361 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     3421 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     3481 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     3541 acaatagcag gcatgctggg gatgcggtcc ggactgtaca attgcattca ttttatgttt
     3601 caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     3661 aaaatcgata aggatcttcc tagagcatgg ctacgtagat aagtagcatg gcgggttaat
     3721 cattaactac aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     3781 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     3841 agtgagcgag cgagcgcgca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3901 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3961 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4021 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4081 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4141 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4201 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4261 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4321 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4381 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4441 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4501 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4561 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4621 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4681 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4741 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4801 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4861 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4921 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4981 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5041 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5101 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5161 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5221 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5281 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5341 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5401 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5461 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5521 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5581 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5641 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5701 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5761 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5821 cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg
     5881 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     5941 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     6001 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatca
//
