LOCUS       sequence-429017-        6805 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP dependent on the combined activity of CRE- and 
            FLP-recombinases.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6805)
  AUTHORS   Rangel Guerrero DK, Balueva K, Barayeu U, Baracskay P, Gridchyn I, 
            Nardin M, Roth CN, Wulff P, Csicsvari J
  TITLE     Hippocampal cholecystokinin-expressing interneurons regulate 
            temporal coding and contextual learning.
  JOURNAL   Neuron. 2024 Apr 10:S0896-6273(24)00197-1. doi: 
            10.1016/j.neuron.2024.03.019.
  PUBMED    38636524
REFERENCE   2  (bases 1 to 6805)
  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: "Neuron. 
            2024 Apr 10:S0896-6273(24)00197-1. doi: 
            10.1016/j.neuron.2024.03.019."
COMMENT     Sequence Label: pAAV-C+F-GFP
FEATURES             Location/Qualifiers
     source          1..6805
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   31..160
                     /label=AAV2 ITR
     promoter        466..743
                     /label=chicken beta-actin promoter
     primer_bind     1022..1044
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1106..1125
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1168..1215
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     intron          1305..1370
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1500..1520
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1945..2079
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1982..2001)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2036..2055
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2145..2192
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     protein_bind    2201..2234
                     /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    2297..2330
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(2337..3050,3051..3053,3054..3056))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2340..3050,3051..3053,3054..3056))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2382..2403)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2731..2750
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2990..3011
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(3088..3121)
                     /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(3192..3225)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3270..3287)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3276..3500
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(3710..4039)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3724..3859
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4024..4044
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(4285..4873)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4365..4384)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5044..5835,5836..5904))
                     /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     5667..5686
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5905..6009)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6077..6095
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6133..6155)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6255..6274
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6488..6507
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6797..6805,1..10)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6797..6805,1..9)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 acactataga atacacggaa ttaattctag ctgcgcgctc gctcgctcac tgaggccgcc
       61 cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg
      121 cgcagagagg gagtggccaa ctccatcact aggggttcct tgtagttaat gattaacccg
      181 ccatgctact tatctacgta gccatgctct aggtaccatt gacgtcaata atgacgtatg
      241 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      301 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      361 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      421 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
      481 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
      541 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg
      601 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc
      661 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
      721 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
      781 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
      841 gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg
      901 tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg
      961 ggagcggctc ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg
     1021 gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc
     1081 ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt
     1141 ggcaaagaat tggatcggcg ccaattggaa gttcctattc cgaagttcct attctctaga
     1201 aagtatagga acttccgatc gctcgaggga tctttgtgaa ggaaccttac ttctgtggtg
     1261 tgacataatt ggacaaacta cctacagaga tttaaagctc taaggtaaat ataaaatttt
     1321 taagtgtata atgtgttaaa ctactgattc taattgtttg tgtattttag attccaacct
     1381 atggaactga tgaatgggag cagtggtgga atgcctttaa tgaggaaaac ctgttttgct
     1441 cagaagaaat gccatctagt gatgatgagg ctactgctga ctctcaacat tctactcctc
     1501 caaaaaagaa gagaaaggta gaagacccca aggactttcc ttcagaattg ctaagttttt
     1561 tgagtcatgc tgtgtttagt aatagaactc ttgcttgctt tgctatttac accacaaagg
     1621 aaaaagctgc actgctatac aagaaaatta tggaaaaata ttctgtaacc tttataagta
     1681 ggcataacag ttataatcat aacatactgt tttttcttac tccacacagg catagagtgt
     1741 ctgctattaa taactatgct caaaaattgt gtacctttag ctttttaatt tgtaaagggg
     1801 ttaataagga atatttgatg tatagtgcct tgactagaga tcataatcag ccataccaca
     1861 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
     1921 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     1981 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     2041 ttgtccaaac tcatcaatgt atcttatcat gtctggatct gacatggtaa gtaagcttgg
     2101 gctgcaggtc gagggaccta acgcgtctcg agtcgcgatt ataagaagtt cctattccga
     2161 agttcctatt ctctagaaag tataggaact tcggatcccc ataacttcgt ataaagtatc
     2221 ctatacgaag ttatatcaaa ataggaagac caatgcttca ccatcgaccc gaattgccaa
     2281 gcatcaccat cgacccataa cttcgtataa tgtatgctat acgaagttat gaattcttac
     2341 ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc cagcaggacc
     2401 atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg actgggtgct caggtagtgg
     2461 ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtcggcg
     2521 agctgcacgc tgccgtcctc gatgttgtgg cggatcttga agttcacctt gatgccgttc
     2581 ttctgcttgt cggccatgat atagacgttg tggctgttgt agttgtactc cagcttgtgc
     2641 cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg gttcaccagg
     2701 gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag
     2761 atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc gtgctgcttc
     2821 atgtggtcgg ggtagcggct gaagcactgc acgccgtagg tcagggtggt cacgagggtg
     2881 ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgtag
     2941 gtggcatcgc cctcgccctc gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc
     3001 agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct caccatggtg
     3061 ggcggccgcc tcgagagatc tgatatcata acttcgtata ggatacttta tacgaagtta
     3121 tcattgggat tcttcctatt ttgatccaag catcaccatc gaccctctag tccagatctc
     3181 accatcgacc cataacttcg tatagcatac attatacgaa gttatgtccc tcgaagaggt
     3241 tcaagcttgg gggtcgactc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     3301 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     3361 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     3421 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3481 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct cgactagagc
     3541 atggctacgt agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga
     3601 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     3661 tcgcccgacg cccgggcggc ctcagtgagc gagcgagcgc gcagagcttt ttgcaaaagc
     3721 ctaggcctcc aaaaaagcct cctcactact tctggaatag ctcagaggcc gaggcggcct
     3781 cggcctctgc ataaataaaa aaaattagtc agccatgggg cggagaatgg gcggaactgg
     3841 gcggagttag gggcgggatg ggcggagtta ggggcgggac tatggttgct gactaattga
     3901 gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca cacctggttg
     3961 ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc
     4021 cacaccctaa ctgacacaca ttccacagct gcattaatga atcggccaac gcgcggggag
     4081 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4141 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4201 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4261 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4321 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4381 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4441 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4501 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4561 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4621 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4681 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4741 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4801 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4861 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4921 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4981 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5041 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5101 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5161 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5221 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5281 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5341 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5401 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5461 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5521 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5581 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5641 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5701 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5761 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5821 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5881 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5941 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6001 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     6061 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga
     6121 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     6181 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     6241 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccatt cgacgctctc
     6301 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     6361 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     6421 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     6481 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     6541 cggccacgat gcgtccggcg tagaggatct ggctagcgat gaccctgctg attggttcgc
     6601 tgaccatttc cgggtgcggg acggcgttac cagaaactca gaaggttcgt ccaaccaaac
     6661 cgactctgac ggcagtttac gagagagatg atagggtctg cttcagtaag ccagatgcta
     6721 cacaattagg cttgtacata ttgtcgttag aacgcggcta caattaatac ataaccttat
     6781 gtatcataca catacgattt aggtg
//
