LOCUS       sequence-464958-        7726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Flp GFP Reporter; FRT-STOP-FRT-GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7726)
  AUTHORS   Tous C, Kinstlinger IS, Rice MEL, Deng J, Wong WW
  TITLE     Multiplexing light-inducible recombinases to control cell fate, 
            Boolean logic, and cell patterning in mammalian cells.
  JOURNAL   Sci Adv. 2025 May 9;11(19):eadt1971. doi: 10.1126/sciadv.adt1971. 
            Epub 2025 May 9.
  PUBMED    40344052
REFERENCE   2  (bases 1 to 7726)
  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: "Sci Adv."; 
            date: "2025-05-9"; volume: "11(19):eadt1971. doi"; pages: " 
            10.1126/sciadv.adt1971. Epub 2025 May 9"
COMMENT     Sequence Label: CT752
FEATURES             Location/Qualifiers
     source          1..7726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(97..116)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      910..1498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1399..1418
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1652..1669
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    1737..1871
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      1891..2025
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     protein_bind    2110..2131
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2146..2151,2152..2169,2170..2176)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2184..2200
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2189..2211
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2208..2224
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2208..2224
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2585..2640
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     CDS             complement(join(2792..3505,3506..3508,3509..3511))
                     /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(2795..3505,3506..3508,3509..3511))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2837..2858)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3186..3205
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3445..3466
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3508..3517
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3562..3595)
                     /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    3612..3746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4086..4141
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     polyA_signal    4452..4676
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(4850..5644)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4961..4980)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5571..5590
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     protein_bind    complement(5687..5720)
                     /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)"
     primer_bind     complement(5768..5787)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          5795..6811
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(5849..5871)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(6812..7088)
                     /label=chicken beta-actin promoter
     enhancer        7090..7469
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7500..7604
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7605..7673,7674..7726,1..739)
                     /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"
ORIGIN
        1 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
       61 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      121 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      181 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
      241 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
      301 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
      361 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
      421 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
      481 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
      541 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
      601 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
      661 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
      721 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
      781 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
      841 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
      901 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
      961 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1021 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     1081 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1141 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1201 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1261 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     1321 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     1381 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     1441 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     1501 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     1561 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     1621 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     1681 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagcggatc
     1741 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     1801 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     1861 ataaacaagt tagctttttg caaaagccta ggcctccaaa aaagcctcct cactacttct
     1921 ggaatagctc agaggccgag gcggcctcgg cctctgcata aataaaaaaa attagtcagc
     1981 catggggcgg agaatgggcg gaactgggcg gagttagggg cgggactatg gttgctgact
     2041 aattgagatg cggatccgct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     2101 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     2161 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     2221 tgaccatgat tacgccaagc ttgggctgca ggtcgaggga tctccataag agaagaggga
     2281 cagctatgac tgggagtagt caggagagga ggaaaaatct ggctagtaaa acatgtaagg
     2341 aaaattttag ggatgttaaa gaaaaaaata acacaaaaca aaatataaaa aaaatctaac
     2401 ctcaagtcaa ggcttttcta tggaataagg aatggacagc agggggctgt ttcatatact
     2461 gatgacctct ttatagccaa cctttgttca tggcagccag catatgggca tatgttgcca
     2521 aactctaaac caaatactca ttctgatgtt ttaaatgatt tgccctccca tatgtccttc
     2581 cgagtgagag acacaaaaaa ttccaacaca ctattgcaat gaaaataaat ttcctttatt
     2641 agccagaagt cagatgctca aggggcttca tgatgtcccc ataatttttg gcagagggaa
     2701 aaagatctca gtggtatttg tgagccaggg cattggccac accagccacc accttctgat
     2761 aggcagcctg cacctgagga gtgcggccgc tttacttgta cagctcgtcc atgccgagag
     2821 tgatcccggc ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt
     2881 ctttgctcag ggcggactgg gtgctcaggt agtggttgtc gggcagcagc acggggccgt
     2941 cgccgatggg ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcgatgt
     3001 tgtggcggat cttgaagttc accttgatgc cgttcttctg cttgtcggcc atgatataga
     3061 cgttgtggct gttgtagttg tactccagct tgtgccccag gatgttgccg tcctccttga
     3121 agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg
     3181 cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg acgtagcctt
     3241 cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc
     3301 actgcacgcc gtaggtcagg gtggtcacga gggtgggcca gggcacgggc agcttgccgg
     3361 tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg ccctcgccgg
     3421 acacgctgaa cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc
     3481 cggtgaacag ctcctcgccc ttgctcacca tggtggcgac cggtggatcc cgggcccgcg
     3541 gtaccgtcga ctgcagaatt cgaagttcct atactttcta gagaatagga acttctcgga
     3601 tttggcgcgc cgatccagac atgataagat acattgatga gtttggacaa accacaacta
     3661 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     3721 ccattataag ctgcaataaa caagttctgc aggtcgaggg atctccataa gagaagaggg
     3781 acagctatga ctgggagtag tcaggagagg aggaaaaatc tggctagtaa aacatgtaag
     3841 gaaaatttta gggatgttaa agaaaaaaat aacacaaaac aaaatataaa aaaaatctaa
     3901 cctcaagtca aggcttttct atggaataag gaatggacag cagggggctg tttcatatac
     3961 tgatgacctc tttatagcca acctttgttc atggcagcca gcatatgggc atatgttgcc
     4021 aaactctaaa ccaaatactc attctgatgt tttaaatgat ttgccctccc atatgtcctt
     4081 ccgagtgaga gacacaaaaa attccaacac actattgcaa tgaaaataaa tttcctttat
     4141 tagccagaag tcagatgctc aaggggcttc atgatgtccc cataattttt ggcagaggga
     4201 aaaagatctc agtggtattt gtgagccagg gcattggcca caccagccac caccttctga
     4261 taggcagcct gcacctgagg agtgcgctag cggtggaagg gctcggagtt gtggtaatct
     4321 atgtatcctg ggcggccgcg actttgcgtg ttgtcttact attgctggca ggaggtcagg
     4381 gtaccgaatt ccgctacagg gcgcgtgggg atacccccta gagccccagc tggttctttc
     4441 cgcctcagaa gccatagagc ccaccgcatc cccagcatgc ctgctattgt cttcccaatc
     4501 ctcccccttg ctgtcctgcc ccaccccacc ccccagaata gaatgacacc tactcagaca
     4561 atgcgatgca atttcctcat tttattagga aaggacagtg ggagtggcac cttccagggt
     4621 caaggaaggc acgggggagg ggcaaacaac agatggctgg caactagaag gcacaggggg
     4681 tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg
     4741 aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat
     4801 ggcaggttgg gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact
     4861 cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca
     4921 cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg
     4981 ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc
     5041 ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct
     5101 cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat
     5161 gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct
     5221 cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc
     5281 gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga
     5341 gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt
     5401 cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt
     5461 cctgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct
     5521 gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat
     5581 agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa
     5641 tcatgcgaaa cgatcctcat cctgtctctt gatcagatca aatccggaag ttcctatact
     5701 ttctagagaa taggaacttc acgcgttcgc tcgaattaat caattctttg ccaaaatgat
     5761 gagacagcac aataaccagc acgttgccca ggagctgtag gaaaaagaag aaggcatgaa
     5821 catggttagc agaggctcta gagccgccgg tcacacgcca gaagccgaac cccgccctgc
     5881 cccgtccccc ccgaaggcag ccgtcccccc gcggacagcc ccgaggctgg agagggagaa
     5941 ggggacggcg gcgcggcgac gcacgaaggc cctccccgcc catttccttc ctgccggcgc
     6001 cgcaccgctt cgccccgcgc ccgctagagg gggtgcggcg gcgcctccca gatttcggct
     6061 ccgcacagat ttgggacaaa ggaagtccct gcgccctctc gcacgattac cataaaaggc
     6121 aatggctgcg gctcgccgcg cctcgacagc cgccggcgct ccgggggccg ccgcgcccct
     6181 cccccgagcc ctccccggcc cgaggcggcc ccgccccgcc cggcaccccc acctgccgcc
     6241 accccccgcc cggcacggcg agccccgcgc cacgccccgt acggagcccc gcacccgaag
     6301 ccgggccgtg ctcagcaact cggggagggg ggtgcagggg gggttgcagc ccgaccgacg
     6361 cgcccacacc ccctgctcac ccccccacgc acacaccccg cacgcagcct ttgttcccct
     6421 cgcagccccc cccgcaccgc ggggcaccgc ccccggccgc gctcccctcg cgcacactgc
     6481 ggagcgcaca aagccccgcg ccgcgcccgc agcgctcaca gccgccgggc agcgcggagc
     6541 cgcacgcggc gctccccacg cacacacaca cgcacgcacc ccccgagccg ctccccccgc
     6601 acaaagggcc ctcccggagc ccctcaaggc tttcacgcag ccacagaaaa gaaacaagcc
     6661 gtcattaaac caagcgctaa ttacagcccg gaggagaagg gccgtcccgc ccgctcacct
     6721 gtgggagtaa cgcggtcagt cagagccggg gcgggcggcg cgaggcggcg gcggagcggg
     6781 gcacggggcg aaggcagcgc gcagcgactc ccgcccgccg cgcgcttcgc tttttatagg
     6841 gccgccgccg ccgccgcctc gccataaaag gaaactttcg gagcgcgccg ctctgattgg
     6901 ctgccgccgc acctctccgc ctcgccccgc cccgcccctc gccccgcccc gccccgcctg
     6961 gcgcgcgccc cccccccccc ccgcccccat cgctgcacaa aataattaaa aaataaataa
     7021 atacaaaatt gggggtgggg agggggggga gatggggaga gtgaagcaga acgtggggct
     7081 cacctcgacc atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc
     7141 ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat
     7201 agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa
     7261 atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt
     7321 cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt
     7381 aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta
     7441 ctattaataa ctagtcaata atcaatgtcg acccaggtgg cacttttcgg ggaaatgtgc
     7501 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     7561 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     7621 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     7681 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacg
//
