LOCUS       sequence-192467-        7275 bp    DNA     circular SYN 15-APR-2025
DEFINITION  To clone gene of interest downstream of FRT1-His-H2B-Cherry-2A 
            cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7275)
  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 7275)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: FRT1-His-H2B-Cherry-2A (IG156)
FEATURES             Location/Qualifiers
     source          1..7275
                     /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"
     regulatory      718..727
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             730..747
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             748..1122
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     CDS             1144..1851
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1289..1307)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1557..1576
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1861..1914
                     /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    1971..2559
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2024..2044)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2442..2453)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2693..2814
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2866..2883)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2872..3096
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3142..3570
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3229..3248)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3439..3460
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3579..3599)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3584..3913
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3764..3899
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3826..3845
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3980..4774
                     /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(4034..4053)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4644..4663
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4948..5069
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5118..5134)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5118..5134)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5131..5153)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5142..5158
                     /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(5166..5172,5173..5190,5191..5196))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5211..5232
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5349..5366)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5520..6108)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5600..5619)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6279..7070,7071..7139))
                     /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     6902..6921
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7140..7244)
                     /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 taagcttgaa gttcctattc ttcaaatagt ataggaactt ccggcgcgcc
      721 accatggcgc atcatcatca tcatcatcca gagccagcga agtctgctcc cgccccgaaa
      781 aagggctcca agaaggcggt gactaaggcg cagaagaaag gcggcaagaa gcgcaagcgc
      841 agccgcaagg agagctattc catctatgtc tacaaggttc tgaagcaggt ccaccctgac
      901 accggcattt cgtccaaggc catgggcatc atgaactcgt ttgtgaacga cattttcgag
      961 cgcatcgcag gtgaggcttc ccgcctggcg cattacaaca agcgctcgac catcacctcc
     1021 agggagatcc agacggccgt gcgcctgctg ctgcctgggg agttggccaa gcacgccgtg
     1081 tccgagggta ctaaggccat caccaagtac accagcgcta aggatccccg ggtaccggta
     1141 accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc
     1201 aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
     1261 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg
     1321 cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag
     1381 caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag
     1441 cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag
     1501 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc
     1561 gtaatgcaga agaagaccat gggctgggag gcctcctccg agagaatgta ccccgaggac
     1621 ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac
     1681 gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac
     1741 gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     1801 gaacgcgccg agggccgcca ctccactggc ggcatggacg agctgtacaa gggcagtgga
     1861 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccagaattc
     1921 tgcagatatc cagcacagtg gcggccgctc gagtctaggc gcgccccgct aatcaacctc
     1981 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     2041 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     2101 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     2161 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     2221 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     2281 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     2341 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     2401 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     2461 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     2521 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt cgagactgct
     2581 gcaccagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
     2641 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     2701 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     2761 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaaggcgt
     2821 ggagatctat cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     2881 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     2941 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3001 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3061 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3121 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3181 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3241 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3301 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3361 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3421 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3481 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3541 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     3601 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3661 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3721 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     3781 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     3841 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     3901 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     3961 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     4021 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     4081 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     4141 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     4201 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     4261 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     4321 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     4381 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     4441 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     4501 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     4561 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     4621 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     4681 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     4741 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     4801 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     4861 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4921 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     4981 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     5041 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     5101 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     5161 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     5221 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5281 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5341 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5401 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5461 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5521 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5581 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5641 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5701 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5761 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5821 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5881 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5941 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6001 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6061 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6121 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6181 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6241 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6301 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6361 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6421 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6481 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6541 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6601 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6661 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6721 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6781 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6841 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6901 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6961 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     7021 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     7081 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7141 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7201 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7261 gtgccacctg acgtc
//
