LOCUS       sequence-394119-        5969 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GCaMP8f in a Cre-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5969)
  AUTHORS   Moffa JC, Bland IN, Tooley JR, Kalyanaraman V, Heitmeier M, Creed 
            MC, Copits BA
  TITLE     Cell-Specific Single Viral Vector CRISPR/Cas9 Editing and 
            Genetically Encoded Tool Delivery in the Central and Peripheral 
            Nervous Systems.
  JOURNAL   eNeuro. 2024 Jul 5;11(7):ENEURO.0438-23.2024. doi: 
            10.1523/ENEURO.0438-23.2024. Print 2024 Jul.
  PUBMED    38871457
REFERENCE   2  (bases 1 to 5969)
  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: 
            "10.1523/ENEURO.0438-23.2024"; journalName: "eNeuro"; date: 
            "2024-07-5- 5"; volume: "11"; issue: "7"; pages: 
            "ENEURO.0438-23.2024"
COMMENT     Sequence Label: pX552-hsyn DIO GCaMP8f(with gRNA scaffold)
FEATURES             Location/Qualifiers
     source          1..5969
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        377..824
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     primer_bind     896..915
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    950..983
                     /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    1046..1079
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1664..1683
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1923..1944
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2050..2071)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(2353..2370)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(2380..2413)
                     /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(2484..2517)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    2572..3048
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2771..2790)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3088..3228
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3088..3217
                     /label=AAV2 ITR
     rep_origin      3303..3758
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3390..3409)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3600..3621
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3775..3794)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3894..3916
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3954..3972)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4040..4144
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4145..4213,4214..5005)
                     /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(4363..4382)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5176..5764
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5665..5684
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5826..5955
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gaagagcgag ctcttctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ctagactgca
      361 gagggccctg cgtatgagtg caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg
      421 cctacctgac gaccgacccc gacccactgg acaagcaccc aacccccatt ccccaaattg
      481 cgcatcccct atcagagagg gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc
      541 cagcttcagc accgcggaca gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc
      601 tcagcactga aggcgcgctg acgtcactcg ccggtccccc gcaaactccc cttcccggcc
      661 accttggtcg cgtccgcgcc gccgccggcc cagccggacc gcaccacgcg aggcgcgaga
      721 taggggggca cgggcgcgac catctgcgct gcggcgccgg cgactcagcg ctgcctcagt
      781 ctgcggtggg cagcggagga gtcgtgtcgt gcctgagagc gcagtcgaga aggtaccgga
      841 tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac
      901 gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tggatcccca taacttcgta
      961 taaagtatcc tatacgaagt tatatcaaaa taggaagacc aatgcttcac catcgacccg
     1021 aattgccaag catcaccatc gacccataac ttcgtataat gtatgctata cgaagttatg
     1081 aattcctgca ggagctcact agtttcactt cgctgtcatc atttgtacaa actcttcgta
     1141 gtttacctga ccatccccat cgatgtctgc ttccctgatc atttcatcaa cctcttcatc
     1201 tgttaacttc tctccaaggt ttgtcatcac gtggcgaagc tctgctgcac tgatgtagcc
     1261 attgccatcc ttatcaaaca caccgaacgc ttctctaatt tcttcttccg tgtccctgta
     1321 tttcattttt cttgccatca ttgtcaggaa ctcagggaag tcgattgtgc cgtcaccgtc
     1381 ggcatctact tcattgatca tgtcctgcag ctctgcttct gtggggttca gtccgagaga
     1441 ccgcatcacc gtccccagct ccttggttgt tattgtccca tccccgtcct tgtcaaatag
     1501 ggagaaagcc tctttaaatt ctgcgatctg ctctccagtc agttggtccg gcaggttgta
     1561 ctccagcttg tgccccagga tgttgccgtc ctccttgaag tcgatgccct tcagctcgat
     1621 gcggttcacc agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc
     1681 gtccttgaag aagatggtgc gctcctggat gtagccttcg ggcatggcgg acttgaagaa
     1741 gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt aggtcagggt
     1801 ggtcacgagg gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt
     1861 cagcttgccg taggtggcat cgccctcacc ctcgccggac acgctgaact tgtggccgtt
     1921 tacgtcgccg tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt
     1981 gctcaccatg ctccctccgg taccgccctt gtacagctcg tccatgccga gagtgatccc
     2041 ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttcga
     2101 aagtttggac tccacgctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat
     2161 gggggtgttc tgctggtagt ggtaggcgag ctgcacgccg ccgtcctcga tgttgtggcg
     2221 gatgtggaag ttcgccttga tgccgttctt ctgcttgtcg gccttgatat agacgttgat
     2281 cttcaggccc atcaggcggg cgatgatctt cacggcggtg gccacctcct tgaaggtctt
     2341 cttgcgacgc gtgtgatgat ggtgatgatg catgatatca taacttcgta taggatactt
     2401 tatacgaagt tatcattggg attcttccta ttttgatcca agcatcacca tcgaccctct
     2461 agtccagatc tcaccatcga cccataactt cgtatagcat acattatacg aagttatgtc
     2521 cctcgaagag gttcaagctt atcgataccg agcgctgctc gagagatcta cgggtggcat
     2581 ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc
     2641 agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat
     2701 attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg
     2761 gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca
     2821 atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc
     2881 caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc
     2941 atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc
     3001 aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag
     3061 gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct
     3121 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     3181 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     3241 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     3301 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3361 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3421 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3481 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     3541 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3601 tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt
     3661 ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt
     3721 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat
     3781 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     3841 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3901 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     3961 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     4021 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4081 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     4141 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     4201 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     4261 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     4321 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     4381 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     4441 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     4501 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     4561 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     4621 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     4681 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     4741 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     4801 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     4861 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     4921 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     4981 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     5041 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     5101 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5161 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5221 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5281 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     5341 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5401 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5461 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5521 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5581 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5641 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5701 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5761 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     5821 catgtcctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg cgtcgggcga
     5881 cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca
     5941 tcactagggg ttcctgcggc cgcacgcgt
//
