LOCUS       sequence-192460-        7707 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To clone gene of interest downstream of FRT2-Mb2Tomato-2A cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7707)
  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 7707)
  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: FRT2-Mb2Tomato-2A (IG154)
FEATURES             Location/Qualifiers
     source          1..7707
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        78..381
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..585
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     536..556
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     630..649
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        630..648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    678..711
                     /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)"
     regulatory      724..733
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             859..2283
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGL
                     VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ
                     ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH
                     LFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYE
                     GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMN
                     FEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVL
                     KGEIHQALKLKDGGRYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYER
                     SEGRHHLFLYGMDELYK"
     CDS             2293..2346
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     misc_feature    2403..2991
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2456..2476)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2874..2885)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3125..3246
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3298..3315)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3304..3528
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3574..4002
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3661..3680)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3871..3892
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4011..4031)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4016..4345
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4196..4331
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4258..4277
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4412..5206
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4466..4485)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5076..5095
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5380..5501
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5550..5566)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5550..5566)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5563..5585)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5574..5590
                     /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(5598..5604,5605..5622,5623..5628))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5643..5664
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5781..5798)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5952..6540)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6032..6051)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6711..7502,7503..7571))
                     /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     7334..7353
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7572..7676)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatcctag ttattaatag taatcaatta cggggtcatt agttcatagc
       61 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      121 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      181 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      241 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      301 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      361 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      421 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      481 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      541 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga
      601 gaacccactg cttactggct tatcgaaatt aatacgactc actataggga gacccaagct
      661 ggctagcaat tagatctgaa gttcctattc tctagaaggt ataggaactt ccggtaccgg
      721 cgcgccacca tgggttgctg tttctccaag accgcagcga agggagaagc cgccgcggag
      781 aggcctgggg aggcggctgt ggcctcgtcg ccttccaaag cgaacggaca ggagaatggc
      841 cacgtgaagg taggatccgt gagcaaggga gaggaggtca tcaaagagtt catgcgcttc
      901 aaggtgcgca tggagggctc catgaacggc cacgagttcg agatcgaggg cgagggcgag
      961 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg cggccccctg
     1021 cccttcgcct gggacatcct gtccccccag ttcatgtacg gctccaaggc gtacgtgaag
     1081 caccccgccg acatccccga ttacaagaag ctgtccttcc ccgagggctt caagtgggag
     1141 cgcgtgatga acttcgagga cggcggtctg gtgaccgtga cccaggactc ctccctgcag
     1201 gacggcacgc tgatctacaa ggtgaagatg cgcggcacca acttcccccc cgacggcccc
     1261 gtaatgcaga agaagaccat gggctgggag gcctccaccg agcgcctgta cccccgcgac
     1321 ggcgtgctga agggcgagat ccaccaggcc ctgaagctga aggacggcgg ccactacctg
     1381 gtggagttca agaccatcta catggccaag aagcccgtgc aactgcccgg ctactactac
     1441 gtggacacca agctggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     1501 gagcgctccg agggccgcca ccacctgttc ctggggcatg gcaccggcag caccggcagc
     1561 ggcagctccg gcaccgcctc ctccgaggac aacaacatgg ccgtcatcaa agagttcatg
     1621 cgcttcaagg tgcgcatgga gggctccatg aacggccacg agttcgagat cgagggcgag
     1681 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggcggc
     1741 cccctgccct tcgcctggga catcctgtcc ccccagttca tgtacggctc caaggcgtac
     1801 gtgaagcacc ccgccgacat ccccgattac aagaagctgt ccttccccga gggcttcaag
     1861 tgggagcgcg tgatgaactt cgaggacggc ggtctggtga ccgtgaccca ggactcctcc
     1921 ctgcaggacg gcacgctgat ctacaaggtg aagatgcgcg gcaccaactt cccccccgac
     1981 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct ccaccgagcg cctgtacccc
     2041 cgcgacggcg tgctgaaggg cgagatccac caggccctga agctgaagga cggcggccgc
     2101 tacctggtgg agttcaagac catctacatg gccaagaagc ccgtgcaact gcccggctac
     2161 tactacgtgg acaccaagct ggacatcacc tcccacaacg aggactacac catcgtggaa
     2221 cagtacgagc gctccgaggg ccgccaccac ctgttcctgt acggcatgga cgagctgtac
     2281 aagggcagtg gagagggcag aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct
     2341 ggcccagaat tctgcagata tccagcacag tggcggccgc tcgagtctag gcgcgccccg
     2401 ctaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     2461 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     2521 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     2581 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     2641 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     2701 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     2761 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
     2821 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     2881 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     2941 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc
     3001 gtcgagactg ctgcaccaga tcataatcag ccataccaca tttgtagagg ttttacttgc
     3061 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     3121 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     3181 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     3241 atcttaaggc gtggactagt atcgagtcta gagggcccgt ttaaacccgc tgatcagcct
     3301 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3361 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3421 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3481 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     3541 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     3601 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3661 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3721 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3781 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3841 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3901 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3961 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     4021 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     4081 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4141 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     4201 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     4261 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     4321 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     4381 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     4441 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     4501 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     4561 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     4621 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     4681 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     4741 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     4801 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     4861 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     4921 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     4981 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     5041 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     5101 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     5161 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     5221 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     5281 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     5341 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     5401 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     5461 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     5521 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     5581 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5641 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5701 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5761 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5821 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5881 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5941 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     6001 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     6061 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     6121 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     6181 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     6241 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     6301 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     6361 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     6421 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6481 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6541 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6601 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6661 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6721 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6781 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6841 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6901 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6961 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     7021 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     7081 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     7141 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     7201 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     7261 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     7321 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     7381 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     7441 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     7501 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7561 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7621 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7681 tttccccgaa aagtgccacc tgacgtc
//
