LOCUS       sequence-192483-        6699 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To clone the 3 ORFs for FlpO dependent generation of ifgMosaics.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6699)
  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 6699)
  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 FRT ifgMosaic Donor (IG135)
FEATURES             Location/Qualifiers
     source          1..6699
                     /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    812..845
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     protein_bind    854..887
                     /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)"
     protein_bind    896..929
                     /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        974..1473
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1009..1032)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1406..1425
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        1485..1532
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             1551..2354
                     /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(1614..1633)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2224..2243
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2386..2403)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2392..2616
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    2773..2894
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2810..2829)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2864..2883
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4478..4494)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4478..4494)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4491..4513)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4502..4518
                     /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(4526..4532,4533..4550,4551..4556))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4571..4592
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4709..4726)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4880..5468)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4960..4979)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5639..6430,6431..6499))
                     /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     6262..6281
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6500..6604)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6672..6690
                     /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 tcctcgagag ggaagttcct attcttcaaa tagtatagga
      841 acttccggcg cgcgaagttc ctattctcta gaaggtatag gaacttcagc gctaggaagt
      901 tcctattctc taaaaagtat aggaacttct acatcgccac gctagcttgg ctgcaggtcg
      961 tcgaaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta
     1021 gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca
     1081 tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact
     1141 cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa
     1201 atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag
     1261 cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag
     1321 aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc
     1381 gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg
     1441 ctgttctcct cttcctcatc tccgggcctt tcgacctgca gcctgttgac aattaatcat
     1501 cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc atgggatcgg
     1561 ccattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     1621 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     1681 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     1741 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     1801 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     1861 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     1921 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     1981 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     2041 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     2101 gcgatgatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     2161 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     2221 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     2281 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     2341 acgagttctt ctgaggggat caattctcta gagctcgctg atcagcctcg actgtgcctt
     2401 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     2461 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     2521 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     2581 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     2641 ggggctcgac tagagcttgc ggaacccttc gtaatcagcc ataccacatt tgtagaggtt
     2701 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacatag aatgaatgca
     2761 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     2821 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     2881 atcaatgtat cttaaggcga agcttcagcc ctatcattcg gcgcgccgcg gccgccacat
     2941 gatggacctg tcgacgtgct ccaaggagag tttgctggca agatcttcgg ggtcgatgct
     3001 aacacaggga atgtgcttgc ctatgagagg ctagaccggg agaaagtctc tgagtacttc
     3061 cttactgccc tcattgtgga caagaacacc aacaaaaacc tggaacaacc ttccagcttc
     3121 actgtcaagg tgcatgatat aaatgacaat tggcctgtgt tttcgcacca ggtattcaac
     3181 gcatctgtgc cagagatgtc agctataggt atgcacctag tcttccctct ctcctccctt
     3241 cctcaacacc agcctgaaga cacctctcac atcagccacc tcaatgctta gggtcaggct
     3301 ccagtaagct caaggaagta gttgtaatgc tatcttgtgc ctttacatga tttcaaaatg
     3361 tgtccctaaa ggctaaactc tgacctagcc ccaaaataaa ccctgaccct aatcatagat
     3421 gggttttaat cttcacaatg tgctgaacac tgactctcat tatagcctgg gccttgatcc
     3481 tgaccgcaga gtgtgctgtc accctagata tgggcttagg aggtactagc ctgtcttcat
     3541 cctgcttaca gaaggtccag gcaaagagtg tgtctctctc tactcttctg tctgggatct
     3601 gtgctcaagg ccatggagcc agaccttctt gaggaaaccc agaactccaa aggatgagaa
     3661 atggaatctc aacccctctg ggaactttgc aacacccttc ctagtcccgc attcatactt
     3721 tcagaaaatg aaaatctacc cagacgtcac ctatgtttct ctctcaaacc caaccccttc
     3781 ccttcttcat ttctcccaca aacacacaca ccacctgttc tctctgcctg cttcatgact
     3841 gctccggctg gcactttgtc ctcatgatgg cccctctagc acaaggcagc cttcattccc
     3901 tcatcttggg cagtaggaac agcagaaccc ccatctactc ccaccaggaa atggggtgag
     3961 ggccagtcat gagcggacat accagaattc tctctctcca cagggacctc tgtcatccgt
     4021 gtgacagcag tggatgcaga tgaccccact gtggccggcc acgccactgt cttgtaccaa
     4081 atcgtgaaag gaaatgagta tttcagcatt gataattctg gtctgtatga ctaagccttt
     4141 tgggcgggta cccatttgga gggatggagg agggacgtga atattgcagg taccagtgta
     4201 tagagaacat taacctgata ggtgtcacca gccactgatc cagagaagga atgcataata
     4261 cacataaaga catgttggag tgggttaggg gagaaggaag cagacaagcg cccaaagcag
     4321 gacctcaagt ccagaaagaa gatcggggcc ttttcaccca agcatagacc aggactcaac
     4381 agaacatgct ggttggaaag tcttttgtac acattcagag acatctgatt tgaacccaga
     4441 actactgctc agcccttaat taagaattcg taatcatgtc atagctgttt cctgtgtgaa
     4501 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4561 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4621 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcga ccaacgcgcg gggagaggcg
     4681 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4741 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4801 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4861 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4921 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4981 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5041 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5101 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5161 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5221 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5281 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5341 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5401 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5461 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5521 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5581 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5641 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5701 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5761 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5821 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5881 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5941 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6001 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6061 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6121 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6181 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6241 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6301 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6361 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6421 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6481 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6541 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6601 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     6661 taacctataa aaataggcgt atcacgaggc cctttcgtc
//
