LOCUS       sequence-436382-        7489 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to generate AAV virus that will express EGFP from the 
            CAG promoter under intersectional control by Flp and Cre 
            recombinases.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7489)
  AUTHORS   Madisen L, Garner AR, Shimaoka D, Chuong AS, Klapoetke NC, Li L, van
            der Bourg A, Niino Y, Egolf L, Monetti C, Gu H, Mills M, Cheng A, 
            Tasic B, Nguyen TN, Sunkin SM, Benucci A, Nagy A, Miyawaki A, 
            Helmchen F, Empson RM, Knopfel T, Boyden ES, Reid RC, Carandini M, 
            Zeng H
  TITLE     Transgenic mice for intersectional targeting of neural sensors and 
            effectors with high specificity and performance.
  JOURNAL   Neuron. 2015 Mar 4;85(5):942-58. doi: 10.1016/j.neuron.2015.02.022.
  PUBMED    25741722
REFERENCE   2  (bases 1 to 7489)
  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: 
            "10.1016/j.neuron.2015.02"; journalName: "Neuron"; date: "2015-03-4-
            4"; volume: "85"; issue: "5"; pages: "942-58"
COMMENT     Sequence Label: AAV pCAG-FSF-FLEX-EGFP-WPRE-bGHpA
FEATURES             Location/Qualifiers
     source          1..7489
                     /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"
     enhancer        873..1176
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1178..1456
                     /label=chicken beta-actin promoter
     intron          1457..2473
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2397..2419
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2481..2500
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    2560..2593
                     /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    2639..2687
                     /note="synthetic polyadenylation signal"
     polyA_signal    2696..3172
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2895..2914)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(3207..3226)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3251..3299
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    3459..3492
                     /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(3512..3545)
                     /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(3556..3589)
                     /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(join(3645..4358,4359..4361,4362..4364))
                     /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(3648..4358,4359..4361,4362..4364))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3690..3711)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4039..4058
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4298..4319
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      4361..4370
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4442..4475
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4486..4519
                     /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    4543..5131
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4596..4616)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5014..5025)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5166..5183)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5172..5379
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5402..5542
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5402..5531
                     /label=AAV2 ITR
     rep_origin      5617..6072
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5704..5723)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5914..5935
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6089..6108)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6208..6230
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6268..6286)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6354..6458
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6459..6527,6528..7319)
                     /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(6677..6696)
                     /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 gcgaaacaat tctgcaggaa tctagttatt aatagtaatc aattacgggg
      841 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      901 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      961 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     1021 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     1081 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     1141 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
     1201 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
     1261 ttattttgtg cagcgatggg ggcggggggg gggggggggg cgcgcgccag gcggggcggg
     1321 gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg
     1381 cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg
     1441 aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc
     1501 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
     1561 ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt
     1621 tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg
     1681 ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc
     1741 cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga
     1801 ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg
     1861 ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct
     1921 gcaacccccc ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg
     1981 gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg
     2041 ggtgccgggc ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg
     2101 cccccggagc gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta
     2161 atcgtgcgag agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg
     2221 gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa
     2281 ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca
     2341 gcctcggggc tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt
     2401 cggcttctgg cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt
     2461 ctttttccta cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa
     2521 agaattgatt tgataccgcg ggccccgcgt ggacgcgaag aagttcctat tctctagaaa
     2581 gtataggaac ttccctcagc accatggcta gcggcagcct cggagtttga atagatagaa
     2641 taaaatatct ttattttcat tccatctgtg tgttggtttt ttgtgtgaga tctacgggtg
     2701 gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc
     2761 caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta
     2821 taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg
     2881 tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac
     2941 tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct caggctcccg agttgttggg
     3001 attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt
     3061 caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc
     3121 tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgcctca
     3181 gctaattgag taggggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc
     3241 cgcgctatga cggcaataaa aagacagaat aaaacgcacg ggtgttgggt cgtttgttca
     3301 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
     3361 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     3421 cccagggctc gcagccaacg tcggggcggc aggccgtgga agttcctatt ctctagaaag
     3481 tataggaact tcctcgagat tttcgacgtc gataacttcg tatagcatac attatacgaa
     3541 gttatccgaa tcgcaataac ttcgtatagg atactttata cgaagttatt tggcgcgcca
     3601 aatttaaata acgcaacgta ccttcgaggg ggcccaagga attcttactt gtacagctcg
     3661 tccatgccga gagtgatccc ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc
     3721 ttctcgttgg ggtctttgct cagggcggac tgggtgctca ggtagtggtt gtcgggcagc
     3781 agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg
     3841 ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg
     3901 gccatgatat agacgttgtg gctgttgtag ttgtactcca gcttgtgccc caggatgttg
     3961 ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg
     4021 aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc
     4081 tggacgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg
     4141 tagcggctga agcactgcac gccgtaggtc agggtggtca cgagggtggg ccagggcacg
     4201 ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt ggcatcgccc
     4261 tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg
     4321 atgggcacca ccccggtgaa cagctcctcg cccttgctca ccatggtggc gaattccccc
     4381 aagcttggaa cgtactaacc ttgatagcta gctagttggc gcgccaacct ggccggcctt
     4441 gataacttcg tataatgtat gctatacgaa gttatccgaa tcgcaataac ttcgtataaa
     4501 gtatcctata cgaagttatc gaagatcatc aagcttatcg ataatcaacc tctggattac
     4561 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     4621 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     4681 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     4741 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     4801 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     4861 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     4921 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt tgccacctgg
     4981 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     5041 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     5101 agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct cgagagatcg
     5161 atctgcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     5221 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     5281 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     5341 gggggaggat tgggaagaca atagcaggca tgctggggac acgtgcggac cgagcggccg
     5401 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     5461 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     5521 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     5581 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     5641 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     5701 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     5761 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     5821 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     5881 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     5941 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     6001 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     6061 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     6121 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     6181 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     6241 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     6301 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     6361 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     6421 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     6481 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6541 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6601 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6661 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6721 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6781 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6841 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6901 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6961 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     7021 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     7081 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     7141 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     7201 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     7261 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     7321 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     7381 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     7441 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
