LOCUS       sequence-414074-        6787 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Cre- and Flp-dependent transgene expression (CRE-ON / FLP-ON) of 
            GCaMP6f in neurons under the control of the hSyn1 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6787)
  TITLE     Cre- and Flp-dependent transgene expression (CRE-ON / FLP-ON) of 
            GCaMP6f in neurons under the control of the hSyn1 promoter, based on
            construct from Pouchelon et al Cell Rep Methods. 2022 May 
            26;2(6):10022
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6787)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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            "Unpublished"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-VTKS4-GCaMP6f
FEATURES             Location/Qualifiers
     source          1..6787
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        145..592
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    616..649
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     polyA_signal    695..743
                     /note="synthetic polyadenylation signal"
     polyA_signal    752..1228
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(951..970)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(1263..1282)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1307..1355
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    1515..1548
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    complement(1555..1588)
                     /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(1639..1672)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(2884..2940)
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     regulatory      2952..2961
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(join(2968..2982,2983..2991))
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             complement(3031..3048)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      3054..3063
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3089..3122
                     /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    3173..3206
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3215..3803
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3268..3288)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3686..3697)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3811..4035
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4037..4177
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4037..4166
                     /label=AAV2 ITR
     rep_origin      4252..4707
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4339..4358)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4549..4570
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4724..4743)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4843..4865
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4903..4921)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4989..5093
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5094..5162,5163..5954)
                     /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(5312..5331)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6125..6713
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6614..6633
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6775..6787,1..117)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc gacctttggt cgcccggcct
       61 cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg ggttcctgct
      121 agactgcaga gggccctgcg tatgagtgca agtgggtttt aggaccagga tgaggcgggg
      181 tgggggtgcc tacctgacga ccgaccccga cccactggac aagcacccaa cccccattcc
      241 ccaaattgcg catcccctat cagagagggg gaggggaaac aggatgcggc gaggcgcgtg
      301 cgcactgcca gcttcagcac cgcggacagt gccttcgccc ccgcctggcg gcgcgcgcca
      361 ccgccgcctc agcactgaag gcgcgctgac gtcactcgcc ggtcccccgc aaactcccct
      421 tcccggccac cttggtcgcg tccgcgccgc cgccggccca gccggaccgc accacgcgag
      481 gcgcgagata ggggggcacg ggcgcgacca tctgcgctgc ggcgccggcg actcagcgct
      541 gcctcagtct gcggtgggca gcggaggagt cgtgtcgtgc ctgagagcgc agtcagatat
      601 cgtttaaact ccggagaagt tcctattctc tagaaagtat aggaacttcc ctcagcacca
      661 tggctagcgg cagcctcgga gtttgaatag atagaataaa atatctttat tttcattcca
      721 tctgtgtgtt ggttttttgt gtgagatcta cgggtggcat ccctgtgacc cctccccagt
      781 gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt
      841 aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt
      901 ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga
      961 accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca
     1021 agcgattctc ctgcctcagg ctcccgagtt gttgggattc caggcatgca tgaccaggct
     1081 cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc
     1141 caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc
     1201 gtgaaccact gctcccttcc ctgtccttct gcctcagcta attgagtagg ggggaggcta
     1261 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     1321 cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
     1381 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
     1441 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
     1501 ggcggcaggc cgtggaagtt cctattctct agaaagtata ggaacttcac cggaataact
     1561 tcgtatagga tactttatac gaagttatgc agaatggtag ctggattgta gctgctatta
     1621 gcaatatgaa acctcttaat aacttcgtat agcatacatt atacgaagtt ataacgcgcc
     1681 actagtgaat tcgagctcgg tacctcactt ggctgtcatc atctggacaa attcttcata
     1741 attgacctga ccgtctccat cgatgtcggc ctctcggatc atctcatcca cttcttcatc
     1801 tgtaagtttc tctcccaagt tggtcatcac atgccggagc tcagcagcgc tgatatagcc
     1861 gttaccatcc ttgtcaaaca cgccaaaggc ttctcttatc tcctcctccg tgtctctgta
     1921 tttcatcttt ctagccatca ttgtcaggaa ttcaggaaag tcaatagttc cgtcaccgtc
     1981 tgcatcgact tcattgatca tgtcctgcag ttctgcctct gtcgggtttt gacccaggga
     2041 cctcatgacg gtgcctaatt ccttggtggt gattgtccca tccccgtcct tatcaaacag
     2101 tgagaattcc tctttaaact ctgcaatctg ctcttctgtc agctgatcag gcagattgta
     2161 ctccagcttg tgcccgagta tattgccatc ctccttgaaa tcaatcccct tgagttctat
     2221 tctgttcacc aaggtatccc cctcaaactt cacctccgcc cgcgtcttat agttgccgtc
     2281 atctttgaag aaaatggtgc gctcctgaat gtagccctct ggcatggcag acttaaagaa
     2341 gtcatgctgc ttcatatgat ctgggtagcg actgaagcac tgtacgccat aagtgagcgt
     2401 ggtgacaagg gttggccagg gtactggcaa cttgccagtc gtacaaatga acttcagagt
     2461 cagtttccca tatgtcgcat ctccttctcc ttctcctgac acagagaatt tgtgtccatt
     2521 cacatcgccg tccagctcca caaggatggg gacaacacct gtgaagagtt cctctccttt
     2581 ggacaccatg ctgcccccag tacctccttt gtagagctcg tccatgccta gagtgattcc
     2641 agcagcagtc acaaactcca atagaaccat gtggtccctt ttctcattgg ggtctttact
     2701 cagcttgctc tgaacagaca aatagtggtt gtcaggtaac agaacaggcc catcaccaat
     2761 gggtgtgttc tgctggtagt ggtatgcgag ttgtacccct ccatcttcga tgttgtgccg
     2821 gatcttgaag ttggctttta tgccattctt ctgcttatct gctttgatgt agacattttc
     2881 aagactgctt agtcgcccaa tggccctcac agcgtggcca gtcttgttcc atttacggcg
     2941 agatgagtcc accatggtgg ccaggtcctt gtcgtcatcg tcatataggt ccctgcccat
     3001 ttgttgacca ccagtcatgg aggccattcc atgatggtgg tgatgatggg accccatggt
     3061 ggcaagcttt actagtggtg gcgctagcat aacttcgtat aaagtatcct atacgaagtt
     3121 atttgcctta acccagaaat tatcactgtt attctttaga atggtgcaaa gaataacttc
     3181 gtataatgta tgctatacga agttatggcg cgccaatcaa cctctggatt acaaaatttg
     3241 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     3301 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     3361 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     3421 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     3481 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     3541 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     3601 gtcggggaag ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg
     3661 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     3721 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     3781 ctccctttgg gccgcctccc cgcctggatc ctgtgccttc tagttgccag ccatctgttg
     3841 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     3901 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     3961 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     4021 cggtgggctc tatggcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     4081 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     4141 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     4201 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     4261 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     4321 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     4381 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     4441 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     4501 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     4561 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     4621 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     4681 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     4741 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     4801 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4861 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     4921 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     4981 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5041 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5101 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5161 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5221 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5281 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5341 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5401 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5461 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5521 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5581 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     5641 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     5701 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     5761 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     5821 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     5881 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     5941 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6001 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6061 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6121 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6181 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6241 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6301 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6361 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6421 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6481 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6541 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6601 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6661 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6721 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtcctgca
     6781 ggcagct
//
