LOCUS       sequence-105724-        7215 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Shuttle vector compatible with Flp-mediated (Flp-in) transgene 
            integration that allows for conditional cDNA and Luc expression upon
            integration. The vector contains an FRT site and two mut loxP 
            sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7215)
  AUTHORS   Huijbers IJ, Del Bravo J, Bin Ali R, Pritchard C, Braumuller TM, van
            Miltenburg MH, Henneman L, Michalak EM, Berns A, Jonkers J
  TITLE     Using the GEMM-ESC strategy to study gene function in mouse models.
  JOURNAL   Nat Protoc. 2015 Nov;10(11):1755-85. doi: 10.1038/nprot.2015.114. 
            Epub 2015 Oct 15.
  PUBMED    26492136
REFERENCE   2  (bases 1 to 7215)
  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/nprot.2015.114"; journalName: "Nat Protoc"; date: 
            "2015-11"; volume: "10"; issue: "11"; pages: "1755-85"
COMMENT     Sequence Label: pFrt-invCAG-ires-Luc
FEATURES             Location/Qualifiers
     source          1..7215
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      172..760
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     661..680
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     914..931
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(951..1016,1017..1054)
                     /label=Ad SA
                     /note="adenovirus major late transcript splice acceptor 
                     (Friedrich and Soriano, 1991)"
     polyA_signal    1167..1288
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    1413..1547
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    1658..1792
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(1794..1827)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    join(1863..2436,2437..2439,2440..2448)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2030..2047)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2257..2276
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2449..4101
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(4130..4147)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4136..4343
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(4349..4396)
                     /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)."
     regulatory      4398..4407
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4410..4443
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4479..4498)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          4506..5521
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(4560..4582)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(5522..5798)
                     /label=chicken beta-actin promoter
     enhancer        5800..6179
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6251..6355
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6356..6424,6425..7215,1)
                     /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(6574..6593)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
       61 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
      121 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
      181 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
      241 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
      301 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
      361 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
      421 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
      481 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
      541 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
      601 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
      661 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
      721 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
      781 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
      841 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
      901 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gacctgcagg tagggcgcag
      961 tagtccaggg tttccttgat gatgtcatac ttatcctgtc cctttttttt ccacagctcg
     1021 cggttgagga caaactcttc gcggtctttc cagtatgcag gatcataatc agccatacca
     1081 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     1141 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     1201 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     1261 gtttgtccaa actcatcaat gtatcttatc atgtctggat gctagagatc ataatcagcc
     1321 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     1381 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     1441 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     1501 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatctag cgatcataat
     1561 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     1621 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     1681 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     1741 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcctaccgtt
     1801 cgtatagcat acattatacg aagttatagg ccggcctccc aagatctacc ctgtttaaac
     1861 cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg
     1921 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc
     1981 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa
     2041 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga
     2101 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc
     2161 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc
     2221 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac
     2281 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg
     2341 tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaacg tctaggcccc ccgaaccacg
     2401 gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaaccat ggaagatgcc
     2461 aaaaacatta agaagggccc agcgccattc tacccactcg aagacgggac cgccggcgag
     2521 cagctgcaca aagccatgaa gcgctacgcc ctggtgcccg gcaccatcgc ctttaccgac
     2581 gcacatatcg aggtggacat tacctacgcc gagtacttcg agatgagcgt tcggctggca
     2641 gaagctatga agcgctatgg gctgaataca aaccatcgga tcgtggtgtg cagcgagaat
     2701 agcttgcagt tcttcatgcc cgtgttgggt gccctgttca tcggtgtggc tgtggcccca
     2761 gctaacgaca tctacaacga gcgcgagctg ctgaacagca tgggcatcag ccagcccacc
     2821 gtcgtattcg tgagcaagaa agggctgcaa aagatcctca acgtgcaaaa gaagctaccg
     2881 atcatacaaa agatcatcat catggatagc aagaccgact accagggctt ccaaagcatg
     2941 tacaccttcg tgacttccca tttgccaccc ggcttcaacg agtacgactt cgtgcccgag
     3001 agcttcgacc gggacaaaac catcgccctg atcatgaaca gtagtggcag taccggattg
     3061 cccaagggcg tagccctacc gcaccgcacc gcttgtgtcc gattcagtca tgcccgcgac
     3121 cccatcttcg gcaaccagat catccccgac accgctatcc tcagcgtggt gccatttcac
     3181 cacggcttcg gcatgttcac cacgctgggc tacttgatct gcggctttcg ggtcgtgctc
     3241 atgtaccgct tcgaggagga gctattcttg cgcagcttgc aagactataa gattcaatct
     3301 gccctgctgg tgcccacact atttagcttc ttcgctaaga gcactctcat cgacaagtac
     3361 gacctaagca acttgcacga gatcgccagc ggcggggcgc cgctcagcaa ggaggtaggt
     3421 gaggccgtgg ccaaacgctt ccacctacca ggcatccgcc agggctacgg cctgacagaa
     3481 acaaccagcg ccattctgat cacccccgaa ggggacgaca agcctggcgc agtaggcaag
     3541 gtggtgccct tcttcgaggc taaggtggtg gacttggaca ccggtaagac actgggtgtg
     3601 aaccagcgcg gcgagctgtg cgtccgtggc cccatgatca tgagcggcta cgttaacaac
     3661 cccgaggcta caaacgctct catcgacaag gacggctggc tgcacagcgg cgacatcgcc
     3721 tactgggacg aggacgagca cttcttcatc gtggaccggc tgaagagcct gatcaaatac
     3781 aagggctacc aggtagcccc agccgaactg gagagcatcc tgctgcaaca ccccaacatc
     3841 ttcgacgccg gggtcgccgg cctgcccgac gacgatgccg gcgagctgcc cgccgcagtc
     3901 gtcgtgctgg aacacggtaa aaccatgacc gagaaggaga tcgtggacta tgtggccagc
     3961 caggttacaa ccgccaagaa gctgcgcggt ggtgttgtgt tcgtggacga ggtgcctaaa
     4021 ggactgaccg gcaagttgga cgcccgcaag atccgcgaga ttctcattaa ggccaagaag
     4081 ggcggcaaga tcgccgtgta gctagcggcc gctagagctc gctgatcagc ctcgactgtg
     4141 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     4201 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     4261 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     4321 gacaatagca ggcatgctgg ggatcgatga agttcctata ctttctagag aataggaact
     4381 tcggaatagt aacttctcca tggtggcggt accgttcgta taatgtatgc tatacgaagt
     4441 tatggcgcgc cgaattcttt gccaaaatga tgagacagca caataaccag cacgttgccc
     4501 aggagctgta ggaaaaagaa gaaggcatga acatggttag cagaggctct agagccgccg
     4561 gtcacacgcc agaagccgaa ccccgccctg ccccgtcccc cccgaaggca gccgtccccc
     4621 cgcggacagc cccgaggctg gagagggaga aggggacggc ggcgcggcga cgcacgaagg
     4681 ccctccccgc ccatttcctt cctgccggcg ccgcaccgct tcgccccgcg cccgctagag
     4741 ggggtgcggc ggcgcctccc agatttcggc tccgcacaga tttgggacaa aggaagtccc
     4801 tgcgccctct cgcacgatta ccataaaagg caatggctgc ggctcgccgc gcctcgacag
     4861 ccgccggcgc tccgggggcc gccgcgcccc tcccccgagc cctccccggc ccgaggcggc
     4921 cccgccccgc ccggcacccc cacctgccgc caccccccgc ccggcacggc gagccccgcg
     4981 ccacgccccg tacggagccc cgcacccgaa gccgggccgt gctcagcaac tcggggaggg
     5041 gggtgcaggg ggggttgcag cccgaccgac gcgcccacac cccctgctca cccccccacg
     5101 cacacacccc gcacgcagcc tttgttcccc tcgcagcccc cccgcaccgc ggggcaccgc
     5161 ccccggccgc gctcccctcg cgcacactgc ggagcgcaca aagccccgcg ccgcgcccgc
     5221 agcgctcaca gccgccgggc agcgcggagc cgcacgcggc gctccccacg cacacacaca
     5281 cgcacgcacc ccccgagccg ctccccccgc acaaagggcc ctcccggagc ccctcaaggc
     5341 tttcacgcag ccacagaaaa gaaacaagcc gtcattaaac caagcgctaa ttacagcccg
     5401 gaggagaagg gccgtcccgc ccgctcacct gtgggagtaa cgcggtcagt cagagccggg
     5461 gcgggcggcg cgaggcggcg gcggagcggg gcacggggcg aaggcagcgc gcagcgactc
     5521 ccgcccgccg cgcgcttcgc tttttatagg gccgccgccg ccgccgcctc gccataaaag
     5581 gaaactttcg gagcgcgccg ctctgattgg ctgccgccgc acctctccgc ctcgccccgc
     5641 cccgcccctc gccccgcccc gccccgcctg gcgcgcgccc cccccccccc ccgcccccat
     5701 cgctgcacaa aataattaaa aaataaataa atacaaaatt gggggtgggg agggggggga
     5761 gatggggaga gtgaagcaga acgtggggct cacctcgacc atggtaatag cgatgactaa
     5821 tacgtagatg tactgccaag taggaaagtc ccataaggtc atgtactggg cataatgcca
     5881 ggcgggccat ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg
     5941 atgtactgcc aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt
     6001 ccctattggc gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt
     6061 gggcggtcag ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg
     6121 gctatgaact aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtca
     6181 acgcgtatat ctggcccgta catcgttaat taagcttatt taaataggtg gcacttttcg
     6241 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     6301 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     6361 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     6421 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     6481 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     6541 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     6601 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     6661 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     6721 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     6781 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6841 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6901 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6961 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     7021 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     7081 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     7141 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     7201 gattaagcat tggta
//
