LOCUS       sequence-192523-        6137 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To clone the 3 ORFs for Tet system (rtTA + Dox) dependent generation
            of ifgMosaics.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6137)
  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 6137)
  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: Tre-Tight ifgMosaic Donor vector (LH121)
FEATURES             Location/Qualifiers
     source          1..6137
                     /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"
     promoter        412..726
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    420..438
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    456..474
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    491..509
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    527..545
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    563..581
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    598..616
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    634..652
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     699..723
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(763..796)
                     /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(804..837)
                     /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(846..879)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      893..902
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             938..958
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             959..1663
                     /codon_start=1
                     /product="Phialidium yellow fluorescent proteim"
                     /label=PhiYFP
                     /note="mammalian codon-optimized"
                     /translation="MSSGALLFHGKIPYVVEMEGNVDGHTFSIRGKGYGDASVGKVDAQ
                     FICTTGDVPVPWSTLVTTLTAGAQCFAKYGPELKDFYKSCMPDGYVQERTITFEGDGNF
                     KTRAEVTFENGSVYNRVKLNGQGFKKDGHVLGKNLEFNFTPHCLYIWGDQANHGLKSAF
                     KICHEITGSKGDFIVADHTQMNTPIGGGPVHVPEYHHMSYHVKLSKDVTDHRDNMSLKE
                     TVRAVDCRKTYL"
     polyA_signal    1846..1967
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1883..1902)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1937..1956
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2031..2064
                     /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        2128..2627
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2163..2186)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2560..2579
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2639..2686
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2705..3508
                     /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(2768..2787)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3378..3397
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3540..3557)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3546..3770
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3827..3860
                     /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(3916..3932)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3916..3932)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3929..3951)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3940..3956
                     /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(3964..3970,3971..3988,3989..3994))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4009..4030
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4147..4164)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4318..4906)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4398..4417)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5077..5868,5869..5937))
                     /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     5700..5719
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5938..6042)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6110..6128
                     /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 cgggcccttt cgtcttcact cgagtttact
      421 ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgatg
      481 tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat
      541 agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttatcc
      601 ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt
      661 cgaggtaggc gtgtacggtg ggaggcctat ataagcagag ctcgtttagt gaaccgtcag
      721 atcgcctgga gaaggatcaa gcttggcaaa gaattcctcg agataacttc gtatagtata
      781 ccttatacga agttatcgag ctcataactt cgtataggat actttatacg aagttataac
      841 tcgagataac ttcgtatagc atacattata cgaagttata ccggcagatc tcgccaccat
      901 ggtgagtgtg attacagagg ctagcttcac catggctcct aagaagaaga ggaaggtgat
      961 gagcagcggc gccctgctgt tccacggcaa gatcccctac gtggtggaga tggagggcaa
     1021 tgtggatggc cacaccttca gcatccgcgg caagggctac ggcgatgcca gcgtgggcaa
     1081 ggtggatgcc cagttcatct gcaccacagg cgatgtgccc gtgccctgga gcaccctggt
     1141 gaccaccctg accgcaggcg cccagtgctt cgccaagtac ggccccgagc tgaaggattt
     1201 ctacaagagc tgcatgcccg atggctacgt gcaggagcgc accatcacct tcgagggtga
     1261 tggcaatttc aagacccgcg ccgaggtgac cttcgagaat ggcagcgtgt acaatcgcgt
     1321 gaagctgaat ggccagggct tcaagaagga tggccacgtg ctgggcaaga atctggagtt
     1381 caatttcacc ccccactgcc tgtacatctg gggcgatcag gccaatcacg gcctgaagag
     1441 cgccttcaag atctgccacg agatcaccgg cagcaagggc gatttcatcg tggccgatca
     1501 cacccagatg aataccccca tcggcggcgg ccccgtgcac gtgcccgagt accaccacat
     1561 gagctaccac gtgaagctga gcaaggatgt gaccgatcac cgcgataata tgagcctgaa
     1621 ggagaccgtg cgcgccgtgg attgccgcaa gacctacctg tgacgcttaa ttagctgagc
     1681 ttggactcct gttgatagat ccagtaatga cctcagaact ccatctggcc gcgactctag
     1741 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     1801 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     1861 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     1921 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg cgaagcttca
     1981 gccctatcat tcggcgcgcc gcggccgcag catagggatc cccgaattcc gaagttccta
     2041 ttctctagaa agtataggaa cttcaggtct gaagaggagt ttacgtccag ccaagctagc
     2101 ttggctgcag gtcgtcgaaa ttctaccggg taggggaggc gcttttccca aggcagtctg
     2161 gagcatgcgc tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc
     2221 gcacacattc cacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg
     2281 cgccaccttc tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt
     2341 gcaggacgtg acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg
     2401 ctgagcaatg gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg
     2461 ctttctgggc tcagaggctg ggaaggggtg ggtccggggg cgggctcagg ggcgggctca
     2521 ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc
     2581 gcacgtctgc cgcgctgttc tcctcttcct catctccggg cctttcgacc tgcagcctgt
     2641 tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta
     2701 aaccatggga tcggccattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     2761 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     2821 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     2881 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     2941 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     3001 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     3061 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     3121 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     3181 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     3241 gcgcatgccc gacggcgatg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     3301 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     3361 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     3421 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     3481 ctatcgcctt cttgacgagt tcttctgagg ggatcaattc tctagagctc gctgatcagc
     3541 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     3601 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     3661 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     3721 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     3781 ggaaagaacc agctggggct cgactagagc ttgcggaacc cttcgcgaag ttcatattct
     3841 ctagaaagta taggaacttc atcagtcagg tacataatat aacttcgggc gcgttaatta
     3901 agaattcgta atcatgtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     3961 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4021 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4081 gctgcattaa tgaatcgacc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4141 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4201 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4261 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4321 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4381 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4441 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4501 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4561 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4621 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4681 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4741 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4801 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4861 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4921 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4981 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5041 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5101 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5161 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5221 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5281 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5341 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5401 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5461 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5521 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5581 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5641 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5701 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5761 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5821 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5881 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5941 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6001 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6061 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     6121 cacgaggccc tttcgtc
//
