LOCUS       sequence-192519-        7658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To clone the 3 ORFs for Cre dependent generation of ifgMosaics.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7658)
  AUTHORS   Pontes-Quero S, Heredia L, Casquero-Garcia V, Fernandez-Chacon M, 
            Luo W, Hermoso A, Bansal M, Garcia-Gonzalez I, Sanchez-Munoz MS, 
            Perea JR, Galiana-Simal A, Rodriguez-Arabaolaza I, Del Olmo-Cabrera 
            S, Rocha SF, Criado-Rodriguez LM, Giovinazzo G, Benedito R
  TITLE     Dual ifgMosaic: A Versatile Method for Multispectral and 
            Combinatorial Mosaic Gene-Function Analysis.
  JOURNAL   Cell. 2017 Aug 10;170(4):800-814.e18. doi: 
            10.1016/j.cell.2017.07.031.
  PUBMED    28802047
REFERENCE   2  (bases 1 to 7658)
  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.2017.07"; journalName: "Cell"; date: "2017-08-10- 
            10"; volume: "170"; issue: "4"; pages: "800-814.e18"
COMMENT     Sequence Label: Triple ORF LoxP ifgMosaic Donor (LH208)
FEATURES             Location/Qualifiers
     source          1..7658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     672..694
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     756..775
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(809..842)
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     protein_bind    complement(850..883)
                     /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)."
     protein_bind    complement(892..925)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      939..948
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             984..1004
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1005..1709
                     /codon_start=1
                     /product="Phialidium yellow fluorescent proteim"
                     /label=PhiYFP
                     /note="mammalian codon-optimized"
                     /translation="MSSGALLFHGKIPYVVEMEGNVDGHTFSIRGKGYGDASVGKVDAQ
                     FICTTGDVPVPWSTLVTTLTAGAQCFAKYGPELKDFYKSCMPDGYVQERTITFEGDGNF
                     KTRAEVTFENGSVYNRVKLNGQGFKKDGHVLGKNLEFNFTPHCLYIWGDQANHGLKSAF
                     KICHEITGSKGDFIVADHTQMNTPIGGGPVHVPEYHHMSYHVKLSKDVTDHRDNMSLKE
                     TVRAVDCRKTYL"
     polyA_signal    1892..2013
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1929..1948)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1983..2002
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2077..2110
                     /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)"
     promoter        2174..2673
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2209..2232)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2606..2625
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2685..2732
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2751..3554
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2814..2833)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3424..3443
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3586..3603)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3592..3816
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3874..3907
                     /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(5437..5453)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5437..5453)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5450..5472)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5461..5477
                     /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)."
     promoter        complement(join(5485..5491,5492..5509,5510..5515))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5530..5551
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5668..5685)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5839..6427)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5919..5938)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6598..7389,7390..7458))
                     /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     7221..7240
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7459..7563)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7631..7649
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgccgg cggctgtcga ggcgcggcga gccgcagcca
      421 ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc aaatctgtgc
      481 ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg cgaagcggtg
      541 cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc
      601 ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc gggggggacg
      661 gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca
      721 tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc
      781 tcatcatttt ggcaaagaat tcctcgagat aacttcgtat agtatacctt atacgaagtt
      841 atcgagctca taacttcgta taggatactt tatacgaagt tataactcga gataacttcg
      901 tatagcatac attatacgaa gttataccgg cagatctcgc caccatggtg agtgtgatta
      961 cagaggctag cttcaccatg gctcctaaga agaagaggaa ggtgatgagc agcggcgccc
     1021 tgctgttcca cggcaagatc ccctacgtgg tggagatgga gggcaatgtg gatggccaca
     1081 ccttcagcat ccgcggcaag ggctacggcg atgccagcgt gggcaaggtg gatgcccagt
     1141 tcatctgcac cacaggcgat gtgcccgtgc cctggagcac cctggtgacc accctgaccg
     1201 caggcgccca gtgcttcgcc aagtacggcc ccgagctgaa ggatttctac aagagctgca
     1261 tgcccgatgg ctacgtgcag gagcgcacca tcaccttcga gggtgatggc aatttcaaga
     1321 cccgcgccga ggtgaccttc gagaatggca gcgtgtacaa tcgcgtgaag ctgaatggcc
     1381 agggcttcaa gaaggatggc cacgtgctgg gcaagaatct ggagttcaat ttcacccccc
     1441 actgcctgta catctggggc gatcaggcca atcacggcct gaagagcgcc ttcaagatct
     1501 gccacgagat caccggcagc aagggcgatt tcatcgtggc cgatcacacc cagatgaata
     1561 cccccatcgg cggcggcccc gtgcacgtgc ccgagtacca ccacatgagc taccacgtga
     1621 agctgagcaa ggatgtgacc gatcaccgcg ataatatgag cctgaaggag accgtgcgcg
     1681 ccgtggattg ccgcaagacc tacctgtgac gcttaattag ctgagcttgg actcctgttg
     1741 atagatccag taatgacctc agaactccat ctggccgcga ctctagatca taatcagcca
     1801 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     1861 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     1921 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     1981 ttgtggtttg tccaaactca tcaatgtatc ttaaggcgaa gcttcagccc tatcattcgg
     2041 cgcgccgcgg ccgcagcata gggatccccg aattccgaag ttcctattct ctagaaagta
     2101 taggaacttc aggtctgaag aggagtttac gtccagccaa gctagcttgg ctgcaggtcg
     2161 tcgaaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta
     2221 gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca
     2281 tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact
     2341 cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa
     2401 atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag
     2461 cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag
     2521 aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc
     2581 gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg
     2641 ctgttctcct cttcctcatc tccgggcctt tcgacctgca gcctgttgac aattaatcat
     2701 cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc atgggatcgg
     2761 ccattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     2821 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     2881 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     2941 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     3001 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     3061 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     3121 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     3181 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     3241 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     3301 gcgatgatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     3361 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     3421 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     3481 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     3541 acgagttctt ctgaggggat caattctcta gagctcgctg atcagcctcg actgtgcctt
     3601 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3661 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3721 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3781 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3841 ggggctcgac tagagcttgc ggaacccttc gttgaagttc ctattctcta gaaagtatag
     3901 gaacttcagt cgacgtgctc caaggagagt ttgctggcaa gatcttcggg gtcgatgcta
     3961 acacagggaa tgtgcttgcc tatgagaggc tagaccggga gaaagtctct gagtacttcc
     4021 ttactgccct cattgtggac aagaacacca acaaaaacct ggaacaacct tccagcttca
     4081 ctgtcaaggt gcatgatata aatgacaatt ggcctgtgtt ttcgcaccag gtattcaacg
     4141 catctgtgcc agagatgtca gctataggta tgcacctagt cttccctctc tcctcccttc
     4201 ctcaacacca gcctgaagac acctctcaca tcagccacct caatgcttag ggtcaggctc
     4261 cagtaagctc aaggaagtag ttgtaatgct atcttgtgcc tttacatgat ttcaaaatgt
     4321 gtccctaaag gctaaactct gacctagccc caaaataaac cctgacccta atcatagatg
     4381 ggttttaatc ttcacaatgt gctgaacact gactctcatt atagcctggg ccttgatcct
     4441 gaccgcagag tgtgctgtca ccctagatat gggcttagga ggtactagcc tgtcttcatc
     4501 ctgcttacag aaggtccagg caaagagtgt gtctctctct actcttctgt ctgggatctg
     4561 tgctcaaggc catggagcca gaccttcttg aggaaaccca gaactccaaa ggatgagaaa
     4621 tggaatctca acccctctgg gaactttgca acacccttcc tagtcccgca ttcatacttt
     4681 cagaaaatga aaatctaccc agacgtcacc tatgtttctc tctcaaaccc aaccccttcc
     4741 cttcttcatt tctcccacaa acacacacac cacctgttct ctctgcctgc ttcatgactg
     4801 ctccggctgg cactttgtcc tcatgatggc ccctctagca caaggcagcc ttcattccct
     4861 catcttgggc agtaggaaca gcagaacccc catctactcc caccaggaaa tggggtgagg
     4921 gccagtcatg agcggacata ccagaattct ctctctccac agggacctct gtcatccgtg
     4981 tgacagcagt ggatgcagat gaccccactg tggccggcca cgccactgtc ttgtaccaaa
     5041 tcgtgaaagg aaatgagtat ttcagcattg ataattctgg tctgtatgac taagcctttt
     5101 gggcgggtac ccatttggag ggatggagga gggacgtgaa tattgcaggt accagtgtat
     5161 agagaacatt aacctgatag gtgtcaccag ccactgatcc agagaaggaa tgcataatac
     5221 acataaagac atgttggagt gggttagggg agaaggaagc agacaagcgc ccaaagcagg
     5281 acctcaagtc cagaaagaag atcggggcct tttcacccaa gcatagacca ggactcaaca
     5341 gaacatgctg gttggaaagt cttttgtaca cattcagaga catctgattt gaacccagaa
     5401 ctactgctca gcccttaatt aagaattcgt aatcatgtca tagctgtttc ctgtgtgaaa
     5461 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     5521 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5581 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcgac caacgcgcgg ggagaggcgg
     5641 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5701 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5761 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5821 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5881 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5941 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     6001 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6061 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6121 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6181 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6241 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6301 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6361 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6421 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6481 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6541 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6601 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6661 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6721 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6781 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6841 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6901 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6961 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     7021 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7081 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7141 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7201 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7261 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7321 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7381 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7441 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7501 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     7561 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     7621 aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
