LOCUS       sequence-458907-        6625 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-off/Flp-on EYFP under the Ef1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6625)
  AUTHORS   Fenno LE, Mattis J, Ramakrishnan C, Hyun M, Lee SY, He M, Tucciarone
            J, Selimbeyoglu A, Berndt A, Grosenick L, Zalocusky KA, Bernstein H,
            Swanson H, Perry C, Diester I, Boyce FM, Bass CE, Neve R, Huang ZJ, 
            Deisseroth K
  TITLE     Targeting cells with single vectors using multiple-feature Boolean 
            logic.
  JOURNAL   Nat Methods. 2014 Jul;11(7):763-72. doi: 10.1038/nmeth.2996. Epub 
            2014 Jun 8.
  PUBMED    24908100
REFERENCE   2  (bases 1 to 6625)
  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: 
            "10.1038/nmeth.2996"; journalName: "Nat Methods"; date: "2014-07"; 
            volume: "11"; issue: "7"; pages: "763-72"
COMMENT     Sequence Label: pAAV-Ef1a-Coff/Fon-EYFP
FEATURES             Location/Qualifiers
     source          1..6625
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        881..2059
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1112..2050
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2007..2027
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(2066..2099)
                     /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(2150..2183)
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     regulatory      2190..2199
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2241..2262)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2375..2408
                     /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    2459..2492
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     protein_bind    2499..2532
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     CDS             complement(2628..2879)
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
                     YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer_bind     complement(2670..2691)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3019..3038
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(3260..3293)
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     misc_feature    3402..3990
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3455..3475)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3873..3884)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4022..4498
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4221..4240)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4538..4678
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4538..4667
                     /label=AAV2 ITR
     rep_origin      4753..5208
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4840..4859)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5050..5071
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5225..5244)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5344..5366
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5404..5422)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5490..5594
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5595..5663,5664..6455)
                     /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(5813..5832)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga ggaatctttg
      841 cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg
      901 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
      961 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
     1021 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
     1081 ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc
     1141 tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta
     1201 cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc
     1261 ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg
     1321 cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat
     1381 ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc
     1441 gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg
     1501 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg
     1561 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat
     1621 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg
     1681 gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg
     1741 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg
     1801 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag
     1861 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg
     1921 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct
     1981 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct
     2041 tccatttcag gtgtcgtgag gatccataac ttcgtatagg atactttata cgaagttatg
     2101 tagctggagc aatttgaaaa gattgttgta ttacctctag ctgcgcttaa taacttcgta
     2161 tagtatacct tatacgaagt tatactagtg ccaccatggt gagcaagggc gaggagctgt
     2221 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     2281 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     2341 gcaccaccgg caaggtgagt acaggagggc tagcataact tcgtataaag tatcctatac
     2401 gaagttattt gccttaaccc agaaattatc actgttattc tttagatagg tgcaaagaat
     2461 aacttcgtat aaggtatact atacgaagtt atggtaccga agttcctatt cttcaaatag
     2521 tataggaact tcaattgtta ttggtgcaaa gaatcacttc cttaagtctt tatactgccc
     2581 aggaagttcc tattcttcaa aaggtatagg aacttcggcg cgccttactt gtacagctcg
     2641 tccatgccga gagtgatccc ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc
     2701 ttctcgttgg ggtctttgct cagggcggac tggtagctca ggtagtggtt gtcgggcagc
     2761 agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg
     2821 ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg
     2881 gccatgatat agacgttgtg gctgttgtag ttgtactcca gcttgtgccc caggatgttg
     2941 ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg
     3001 aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc
     3061 tggacgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg
     3121 tagcgggcga agcactgcag gccgtagccg aaggtggtca cgagggtggg ccagggcacg
     3181 ggcagctgtg ggacgacatg acttaaccag ttaaccatgt tctctgaaca tgagaagggc
     3241 tggccactct ccatctagag aagttcctat actatttgaa gaataggaac ttcttgcctt
     3301 aacccagaaa ttatcactgt tattctttag aatagtgcaa agagaagttc ctataccttt
     3361 tgaagaatag gaacttcgaa ttcgatatca agcttatcga taatcaacct ctggattaca
     3421 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     3481 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     3541 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     3601 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     3661 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     3721 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     3781 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctatgtt gccacctgga
     3841 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     3901 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     3961 gtcggatctc cctttgggcc gcctccccgc atcgataccg agcgctgctc gagagatcta
     4021 cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc
     4081 agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc
     4141 cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac
     4201 aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg
     4261 gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt
     4321 gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg
     4381 gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc
     4441 ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct
     4501 gattttgtag gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg
     4561 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     4621 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     4681 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     4741 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4801 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     4861 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4921 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     4981 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     5041 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     5101 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     5161 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     5221 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     5281 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5341 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     5401 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     5461 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5521 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5581 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5641 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5701 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5761 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5821 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5881 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5941 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     6001 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     6061 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6121 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6181 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6241 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6301 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6361 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6421 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6481 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6541 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6601 ccgtagaaaa gatcaaagga tcttc
//
