LOCUS       sequence-105728-        6503 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Shuttle vector compatible with Flp-mediated (Flp-in) transgene 
            integration that allows for conditional Luciferase expression upon 
            integration. The vector contains an FRT site and two mutant loxP 
            sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6503)
  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 6503)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-Luc
FEATURES             Location/Qualifiers
     source          1..6503
                     /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"
     polyA_signal    1056..1177
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    1302..1436
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    1547..1681
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(1683..1716)
                     /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)."
     regulatory      1731..1740
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1737..3389
                     /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(3418..3435)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3424..3631
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(3637..3684)
                     /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      3686..3695
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3698..3731
                     /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(3767..3786)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          3794..4809
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(3848..3870)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(4810..5086)
                     /label=chicken beta-actin promoter
     enhancer        5088..5467
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5539..5643
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5644..5712,5713..6503,1)
                     /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(5862..5881)
                     /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 atcataatca
      961 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
     1021 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     1081 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     1141 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatg ctagagatca
     1201 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     1261 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     1321 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     1381 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatctagc
     1441 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     1501 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     1561 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     1621 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     1681 cctaccgttc gtatagcata cattatacga agttatggcc ggccagatct gccaccatgg
     1741 aagatgccaa aaacattaag aagggcccag cgccattcta cccactcgaa gacgggaccg
     1801 ccggcgagca gctgcacaaa gccatgaagc gctacgccct ggtgcccggc accatcgcct
     1861 ttaccgacgc acatatcgag gtggacatta cctacgccga gtacttcgag atgagcgttc
     1921 ggctggcaga agctatgaag cgctatgggc tgaatacaaa ccatcggatc gtggtgtgca
     1981 gcgagaatag cttgcagttc ttcatgcccg tgttgggtgc cctgttcatc ggtgtggctg
     2041 tggccccagc taacgacatc tacaacgagc gcgagctgct gaacagcatg ggcatcagcc
     2101 agcccaccgt cgtattcgtg agcaagaaag ggctgcaaaa gatcctcaac gtgcaaaaga
     2161 agctaccgat catacaaaag atcatcatca tggatagcaa gaccgactac cagggcttcc
     2221 aaagcatgta caccttcgtg acttcccatt tgccacccgg cttcaacgag tacgacttcg
     2281 tgcccgagag cttcgaccgg gacaaaacca tcgccctgat catgaacagt agtggcagta
     2341 ccggattgcc caagggcgta gccctaccgc accgcaccgc ttgtgtccga ttcagtcatg
     2401 cccgcgaccc catcttcggc aaccagatca tccccgacac cgctatcctc agcgtggtgc
     2461 catttcacca cggcttcggc atgttcacca cgctgggcta cttgatctgc ggctttcggg
     2521 tcgtgctcat gtaccgcttc gaggaggagc tattcttgcg cagcttgcaa gactataaga
     2581 ttcaatctgc cctgctggtg cccacactat ttagcttctt cgctaagagc actctcatcg
     2641 acaagtacga cctaagcaac ttgcacgaga tcgccagcgg cggggcgccg ctcagcaagg
     2701 aggtaggtga ggccgtggcc aaacgcttcc acctaccagg catccgccag ggctacggcc
     2761 tgacagaaac aaccagcgcc attctgatca cccccgaagg ggacgacaag cctggcgcag
     2821 taggcaaggt ggtgcccttc ttcgaggcta aggtggtgga cttggacacc ggtaagacac
     2881 tgggtgtgaa ccagcgcggc gagctgtgcg tccgtggccc catgatcatg agcggctacg
     2941 ttaacaaccc cgaggctaca aacgctctca tcgacaagga cggctggctg cacagcggcg
     3001 acatcgccta ctgggacgag gacgagcact tcttcatcgt ggaccggctg aagagcctga
     3061 tcaaatacaa gggctaccag gtagccccag ccgaactgga gagcatcctg ctgcaacacc
     3121 ccaacatctt cgacgccggg gtcgccggcc tgcccgacga cgatgccggc gagctgcccg
     3181 ccgcagtcgt cgtgctggaa cacggtaaaa ccatgaccga gaaggagatc gtggactatg
     3241 tggccagcca ggttacaacc gccaagaagc tgcgcggtgg tgttgtgttc gtggacgagg
     3301 tgcctaaagg actgaccggc aagttggacg cccgcaagat ccgcgagatt ctcattaagg
     3361 ccaagaaggg cggcaagatc gccgtgtagc tagcggccgc tagagctcgc tgatcagcct
     3421 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3481 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3541 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3601 attgggaaga caatagcagg catgctgggg atcgatgaag ttcctatact ttctagagaa
     3661 taggaacttc ggaatagtaa cttctccatg gtggcggtac cgttcgtata atgtatgcta
     3721 tacgaagtta tggcgcgccg aattctttgc caaaatgatg agacagcaca ataaccagca
     3781 cgttgcccag gagctgtagg aaaaagaaga aggcatgaac atggttagca gaggctctag
     3841 agccgccggt cacacgccag aagccgaacc ccgccctgcc ccgtcccccc cgaaggcagc
     3901 cgtccccccg cggacagccc cgaggctgga gagggagaag gggacggcgg cgcggcgacg
     3961 cacgaaggcc ctccccgccc atttccttcc tgccggcgcc gcaccgcttc gccccgcgcc
     4021 cgctagaggg ggtgcggcgg cgcctcccag atttcggctc cgcacagatt tgggacaaag
     4081 gaagtccctg cgccctctcg cacgattacc ataaaaggca atggctgcgg ctcgccgcgc
     4141 ctcgacagcc gccggcgctc cgggggccgc cgcgcccctc ccccgagccc tccccggccc
     4201 gaggcggccc cgccccgccc ggcaccccca cctgccgcca ccccccgccc ggcacggcga
     4261 gccccgcgcc acgccccgta cggagccccg cacccgaagc cgggccgtgc tcagcaactc
     4321 ggggaggggg gtgcaggggg ggttgcagcc cgaccgacgc gcccacaccc cctgctcacc
     4381 cccccacgca cacaccccgc acgcagcctt tgttcccctc gcagcccccc cgcaccgcgg
     4441 ggcaccgccc ccggccgcgc tcccctcgcg cacactgcgg agcgcacaaa gccccgcgcc
     4501 gcgcccgcag cgctcacagc cgccgggcag cgcggagccg cacgcggcgc tccccacgca
     4561 cacacacacg cacgcacccc ccgagccgct ccccccgcac aaagggccct cccggagccc
     4621 ctcaaggctt tcacgcagcc acagaaaaga aacaagccgt cattaaacca agcgctaatt
     4681 acagcccgga ggagaagggc cgtcccgccc gctcacctgt gggagtaacg cggtcagtca
     4741 gagccggggc gggcggcgcg aggcggcggc ggagcggggc acggggcgaa ggcagcgcgc
     4801 agcgactccc gcccgccgcg cgcttcgctt tttatagggc cgccgccgcc gccgcctcgc
     4861 cataaaagga aactttcgga gcgcgccgct ctgattggct gccgccgcac ctctccgcct
     4921 cgccccgccc cgcccctcgc cccgccccgc cccgcctggc gcgcgccccc cccccccccc
     4981 gcccccatcg ctgcacaaaa taattaaaaa ataaataaat acaaaattgg gggtggggag
     5041 gggggggaga tggggagagt gaagcagaac gtggggctca cctcgaccat ggtaatagcg
     5101 atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat gtactgggca
     5161 taatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact tggcatatga
     5221 tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca ttgacgtcaa
     5281 tggaaagtcc ctattggcgt tactatggga acatacgtca ttattgacgt caatgggcgg
     5341 gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac gcggaactcc
     5401 atatatgggc tatgaactaa tgaccccgta attgattact attaataact agtcaataat
     5461 caatgtcaac gcgtatatct ggcccgtaca tcgttaatta agcttattta aataggtggc
     5521 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5581 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5641 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5701 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5761 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5821 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     5881 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5941 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     6001 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     6061 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     6121 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     6181 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     6241 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     6301 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     6361 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6421 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6481 gcctcactga ttaagcattg gta
//
