LOCUS       sequence-192468-        7297 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To clone gene of interest downstream of FRT3-HA-H2B-Cerulean-2A 
            cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7297)
  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 7297)
  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: FRT3-HA-H2B-Cerulean-2A (IG158))
FEATURES             Location/Qualifiers
     source          1..7297
                     /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      718..727
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             730..756
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             757..1131
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     regulatory      1150..1159
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1156..1158,1159..1161,1162..1872)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1201..1222)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1462..1481)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1809..1830
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1882..1935
                     /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    1992..2580
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2045..2065)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2463..2474)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2714..2835
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2888..2905)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2894..3118
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3164..3592
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3251..3270)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3461..3482
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3601..3621)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3606..3935
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3786..3921
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3848..3867
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4002..4796
                     /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(4056..4075)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4666..4685
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4970..5091
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5140..5156)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5140..5156)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5153..5175)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5164..5180
                     /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(5188..5194,5195..5212,5213..5218))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5233..5254
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5371..5388)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5542..6130)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5622..5641)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6301..7092,7093..7161))
                     /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     6924..6943
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7162..7266)
                     /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 tactagtgaa gttcctattc tctaaaaagt ataggaactt ccggcgcgcc
      721 accatggcgt atccatatga cgtcccagac tatgccccag agccagcgaa gtctgctccc
      781 gccccgaaaa agggctccaa gaaggcggtg actaaggcgc agaagaaagg cggcaagaag
      841 cgcaagcgca gccgcaagga gagctattcc atctatgtct acaaggttct gaagcaggtc
      901 caccctgaca ccggcatttc gtccaaggcc atgggcatca tgaactcgtt tgtgaacgac
      961 attttcgagc gcatcgcagg tgaggcttcc cgcctggcgc attacaacaa gcgctcgacc
     1021 atcacctcca gggagatcca gacggccgtg cgcctgctgc tgcctgggga gttggccaag
     1081 cacgccgtgt ccgagggtac taaggccatc accaagtaca ccagcgctaa ggatccccgg
     1141 gtaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     1201 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     1261 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     1321 gtgccctggc ccaccctcgt gaccaccctg acctggggcg tgcagtgctt cgcccgctac
     1381 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1441 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     1501 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1561 aacatcctgg ggcacaagct ggagtacaac gccatcagcg acaacgtcta tatcaccgcc
     1621 gacaagcaga agaacggcat caaggccaac ttcaagatcc gccacaacat cgaggacggc
     1681 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1741 ctgcccgaca accactacct gagcacccag tccaagctga gcaaagaccc caacgagaag
     1801 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     1861 gagctgtaca agggcagtgg agagggcaga ggaagtctgc taacatgcgg tgacgtcgag
     1921 gagaatcctg gcccagaatt ctgcagatat ccagcacagt ggcggccgct cgagtctagg
     1981 cgcgccccgc taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     2041 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     2101 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     2161 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     2221 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     2281 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     2341 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     2401 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     2461 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     2521 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     2581 atcgataccg tcgagactgc tgcaccagat cataatcagc cataccacat ttgtagaggt
     2641 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
     2701 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     2761 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     2821 catcaatgta tcttaaggcg tggcggccgc atcgagtcta gagggcccgt ttaaacccgc
     2881 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2941 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3001 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     3061 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     3121 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     3181 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3241 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3301 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3361 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3421 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3481 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3541 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     3601 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3661 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3721 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3781 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3841 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     3901 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     3961 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     4021 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     4081 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     4141 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     4201 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     4261 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     4321 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     4381 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     4441 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     4501 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     4561 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4621 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4681 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4741 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4801 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     4861 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4921 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     4981 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     5041 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     5101 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     5161 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     5221 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     5281 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5341 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5401 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5461 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5521 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5581 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5641 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5701 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5761 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5821 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5881 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5941 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     6001 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6061 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6121 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6181 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6241 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6301 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6361 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6421 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6481 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6541 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6601 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6661 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6721 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6781 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6841 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6901 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6961 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7021 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     7081 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     7141 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     7201 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7261 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
