LOCUS       sequence-374535-        7306 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FlpO dependent GFP expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7306)
  AUTHORS   Winnubst J, Bas E, Ferreira TA, Wu Z, Economo MN, Edson P, Arthur 
            BJ, Bruns C, Rokicki K, Schauder D, Olbris DJ, Murphy SD, Ackerman 
            DG, Arshadi C, Baldwin P, Blake R, Elsayed A, Hasan M, Ramirez D, 
            Dos Santos B, Weldon M, Zafar A, Dudman JT, Gerfen CR, Hantman AW, 
            Korff W, Sternson SM, Spruston N, Svoboda K, Chandrashekar J
  TITLE     Reconstruction of 1,000 Projection Neurons Reveals New Cell Types 
            and Organization of Long-Range Connectivity in the Mouse Brain.
  JOURNAL   Cell. 2019 Sep 19;179(1):268-281.e13. doi: 
            10.1016/j.cell.2019.07.042. Epub 2019 Sep 5.
  PUBMED    31495573
REFERENCE   2  (bases 1 to 7306)
  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.cell.2019.07.042"; journalName: "Cell"; date: 
            "2019-09-19- 19"; volume: "179"; issue: "1"; pages: "268-281.e13"
COMMENT     Sequence Label: pAAV-CAG-FRT-Rev-3xGFP
FEATURES             Location/Qualifiers
     source          1..7306
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             12..68
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(69..71,72..782)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             792..845
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(846..848,849..1559)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     protein_bind    complement(1594..1627)
                     /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)"
     protein_bind    complement(1670..1717)
                     /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)."
     primer_bind     complement(1758..1777)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(1839..1861)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     repeat_region   2729..2858
                     /label=AAV2 ITR
     rep_origin      2930..3385
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3017..3036)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3227..3248
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3411..3515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3516..3584,3585..4376)
                     /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(3734..3753)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4547..5135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5036..5055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5372..5501
                     /label=AAV2 ITR
     polyA_signal    5663..5784
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5700..5719)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5754..5773
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    5827..6415
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             5933..5944
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     6342..6362
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    6450..6483
                     /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)"
     protein_bind    6512..6559
                     /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)."
     CDS             join(6592..6594,6595..6597,6598..7306,1..2)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6592..6594,6595..6597,6598..7306,1..2)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
ORIGIN
        1 agggctccgg cgccaccaac ttctccctgc tcaagcaggc cggggacgtg gaagagaacc
       61 ccggacccgt gtcgaaaggg gaggaactgt tcacgggagt ggtgcctatc ctcgtcgaac
      121 tcgacggaga tgtgaacgga cataagttct cggtgtccgg ggaaggagaa ggggacgcta
      181 cttacggaaa gctgactctg aagtttatct gtaccactgg aaagctccca gtcccgtggc
      241 caacccttgt caccaccttg acttacggag tgcaatgttt ctcccggtac ccggaccaca
      301 tgaagcagca cgacttcttc aaatccgcga tgccggaagg atacgtgcag gagcggacaa
      361 tcttctttaa ggacgacgga aactataaga ccagggccga ggtcaagttt gagggggata
      421 ccctcgtgaa cagaattgaa ctgaagggta ttgacttcaa ggaagatggc aacatcttgg
      481 ggcataagct cgaatacaat tacaactccc acaacgtgta cattatggcg gacaaacaga
      541 agaatggtat taaggtcaat ttcaagatca ggcataacat tgaggacgga tcggtgcaat
      601 tggccgatca ctaccaacag aataccccga ttggagatgg gccggtgctc ctgcctgata
      661 accactactt gtccactcag tccgctctgt cgaaggaccc aaacgaaaag cgggatcata
      721 tggtgctcct ggaatttgtc actgcggctg gtattaccct ggggatggac gaactgtaca
      781 aaggtagcgg agaaggccgc gggtcactgc tgacttgcgg cgacgtggag gaaaacccgg
      841 gtcctgtgtc caagggagag gaactcttca ctggagtggt gccgattctg gtggagctgg
      901 atggggacgt gaacggtcac aaattctccg tgtccggaga aggagaggga gatgctacct
      961 acggaaagct tactctcaag ttcatctgca ccaccggaaa gctgccagtg ccgtggccga
     1021 cacttgtgac tacccttacc tacggagtgc aatgcttctc gcggtaccct gaccatatga
     1081 agcaacatga tttctttaag tccgcaatgc ctgaaggata tgtgcaggaa agaactatct
     1141 ttttcaagga tgatggaaac tacaaaactc gggccgaagt caagttcgaa ggagacacgc
     1201 tcgtgaaccg gatcgaactt aagggaattg actttaagga agatggaaac attctcggtc
     1261 acaagctgga gtacaactac aactctcata acgtctacat catggcagac aagcagaaga
     1321 acggtatcaa ggtcaacttc aaaatcaggc acaatattga agatggttca gtccagctgg
     1381 cagaccacta tcagcaaaat actccgatag gagatggtcc tgtgctgctt cctgacaatc
     1441 attacctgtc aactcagtcg gccctgtcaa aggacccgaa cgaaaagaga gatcacatgg
     1501 tgttgctgga attcgtgacg gcggcgggaa tcaccctggg aatggacgaa ttgtacaaat
     1561 gatgaaccgg tacgcgttta attaagttta aacgaagttc ctatactatt tgaagaatag
     1621 gaacttcgga ataggaactt cagagcgctt tggccgctcc ggccaaaggg aagttcctat
     1681 actttctaga gaataggaac ttcggaatag gaacttcggc gcgccggatc caattctttg
     1741 ccaaaatgat gagacagcac aataaccagc acgttgccca ggagctgtag gaaaaagaag
     1801 aaggcatgaa catggttagc agaggctcta gagccgccgg tcacacgcca gaagccgaac
     1861 cccgccctgc cccgtccccc ccgaaggcag ccgtcccccc gcggacagcc ccgagccgct
     1921 ccccccgcac aaagggccct cccggagccc ctcaaggctt tcacgcagcc acagaaaaga
     1981 aacaagccgt cattaaacca agcgctaatt acagcccgga ggagaagggc cgtcccgccc
     2041 gctcacctgt gggagtaacg cggtcagtca gagccggggc gggcggcgcg aggcggcggc
     2101 ggagcggggc acggggcgaa ggcagcgcgc agcgactccc gcccgccgcg cgcttcgctt
     2161 tttatagggc cgccgccgcc gccgcctcgc cataaaagga aactttcgga gcgcgccgct
     2221 ctgattggct gccgccgcac ctcyccsccy csccccsccc ccccccccsc cccccccccc
     2281 ccccccyggc gcgcgccccc cccccccccc gcccccatcg ctgcacaaaa taattaaaaa
     2341 ataaataaat acaaaattgg gggtggggag gggggggaga tggggagagt gaagcagaac
     2401 gtggggctca cctcgaccat ggtaatagcg atgactaata cgtagatgta ctgccaagta
     2461 ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg
     2521 acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt
     2581 taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt tactatggga
     2641 acatacgtca ttattgacgt caatggtacg atcttcctag agcatggcta cgtagataag
     2701 tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc
     2761 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     2821 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct ggcgtaatag cgaagaggcc
     2881 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt
     2941 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3001 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3061 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3121 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     3181 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3241 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     3301 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     3361 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3421 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3481 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3541 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3601 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3661 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3721 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     3781 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3841 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3901 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3961 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     4021 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     4081 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     4141 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     4201 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     4261 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     4321 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     4381 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4441 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4501 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4561 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4621 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4681 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4741 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4801 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4861 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4921 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4981 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5041 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5101 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5161 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     5221 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     5281 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     5341 tccccgcgcg ttggccgatt cattaatgca gctgcgcgct cgctcgctca ctgaggccgc
     5401 ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc
     5461 gcgcagagag ggagtggcca actccatcac taggggttcc ttgtagttaa tgattaaccc
     5521 gccatgctac ttatctacgt agccatgctc taggaagatc cttatcgatt ttaccacatt
     5581 tgtagaggtt ttacttgctt taaaaaacct cccacatctc cccctgaacc tgaaacataa
     5641 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     5701 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     5761 gtccaaactc atcaatgtat cttatcatgt ctgctcgaag cggccgcccg ggtcgacggt
     5821 atcgatgcgg ggaggcggcc caaagggaga tccgactcgt ctgagggcga aggcgaagac
     5881 gcggaagagg ccgcagagcc ggcagcaggc cgcgggaagg aaggtccgct ggattgaggg
     5941 ccgaagggac gtagcagaag gacgtcccgc gcagaatcca ggtggcaaca caggcgagca
     6001 gccaaggaaa ggacgatgat ttccccgaca acaccacgga attgtcagtg cccaacagcc
     6061 gagcccctgt ccagcagcgg gcaaggcagg cggcgatgag ttccgccgtg gcaataggga
     6121 gggggaaagc gaaagtcccg gaaaggagct gacaggtggt ggcaatgccc caaccagtgg
     6181 gggttgcgtc agcaaacaca gtgcacacca cgccacgttg cctgacaacg ggccacaact
     6241 cctcataaag agacagcaac caggatttat acaaggagga gaaaatgaaa gccatacggg
     6301 aagcaatagc atgatacaaa ggcattaaag cagcgtatcc acatagcgta aaaggagcaa
     6361 catagttaag aataccagtc aatctttcac aaattttgta atccagaggt tgattatcga
     6421 taagcttcct gcagggaagt tcctattccg aagttcctat tcttcaaata gtataggaac
     6481 ttcttggatc aaaataggag cgatcgcaat ggaagttcct attccgaagt tcctattctc
     6541 tagaaagtat aggaacttcg tttaaactgg ccggccacta gtctcgagac catggtgagc
     6601 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     6661 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     6721 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     6781 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     6841 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     6901 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     6961 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     7021 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     7081 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     7141 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     7201 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     7261 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtaca
//
