LOCUS       sequence-223852-        6762 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CFP-KRAS4B fusion protein used for FRET studies in 
            mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6762)
  AUTHORS   Ambrogio C, Kohler J, Zhou ZW, Wang H, Paranal R, Li J, Capelletti 
            M, Caffarra C, Li S, Lv Q, Gondi S, Hunter JC, Lu J, Chiarle R, 
            Santamaria D, Westover KD, Janne PA
  TITLE     KRAS Dimerization Impacts MEK Inhibitor Sensitivity and Oncogenic 
            Activity of Mutant KRAS.
  JOURNAL   Cell. 2018 Feb 8;172(4):857-868.e15. doi: 
            10.1016/j.cell.2017.12.020. Epub 2018 Jan 11.
  PUBMED    29336889
REFERENCE   2  (bases 1 to 6762)
  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.2017.12.020"; journalName: "Cell"; date: "2018-02-8-
            8"; volume: "172"; issue: "4"; pages: "857-868.e15"
COMMENT     Sequence Label: pcDNA3.1-CFP-KRAS4B
FEATURES             Location/Qualifiers
     source          1..6762
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        189..568
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        569..772
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     723..743
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     817..836
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        817..835
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(931..933,934..936,937..1647)
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(976..997)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1237..1256)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1584..1605
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1681..2247
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     promoter        complement(2269..2287)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2270..2287)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2307..2324)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2313..2537
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2583..3011
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2670..2689)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2880..2901
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3020..3040)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3025..3354
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3205..3340
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3267..3286
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3421..4215
                     /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(3475..3494)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4085..4104
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4389..4510
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4426..4445)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4480..4499
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4559..4575)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4559..4575)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4572..4594)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4583..4599
                     /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(4607..4613,4614..4631,4632..4637))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4652..4673
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4790..4807)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4961..5549)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5041..5060)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5720..6511,6512..6580))
                     /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     6343..6362
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6581..6685)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(join(6760..6762,1..17))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
       61 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
      121 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
      181 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
      241 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      301 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      361 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      421 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      481 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      541 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      601 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      661 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      721 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct
      781 aactagagaa cccactgctt actggcttat cgaaattaat acgactcact atagggagac
      841 ccaagcttgg taccgagctc ggatccacta gtaacggccg ccagtgtgct ggaattctgc
      901 agatatccat cacactggcg gccgctcgag atggtgagca agggcgagga gctgttcacc
      961 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacag gttcagcgtg
     1021 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1081 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgacctg gggcgtgcag
     1141 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1201 gaaggctacg tccaggagcg taccatcttc ttcaaggacg acggcaacta caagacccgc
     1261 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1321 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacat cagccacaac
     1381 gtctatatca ccgccgacaa gcagaagaac ggcatcaagg cccacttcaa gatccgccac
     1441 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1501 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     1561 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1621 actctcggca tggacgagct gtacaagggc gccggatcca agctggctag cgccgccacc
     1681 atgactgaat ataaacttgt ggtagttgga gctggtggcg taggcaagag tgccttgacg
     1741 atacagctaa ttcagaatca ttttgtggac gaatatgatc caacaataga ggattcctac
     1801 aggaagcaag tagtaattga tggagaaacc tgtctcttgg atattctcga cacagcaggt
     1861 caagaggagt acagtgcaat gagggaccag tacatgagga ctggggaggg ctttctttgt
     1921 gtatttgcca taaataatac taaatcattt gaagatattc accattatag agaacaaatt
     1981 aaaagagtta aggactctga agatgtacct atggtcctag taggaaataa atgtgatttg
     2041 ccttctagaa cagtagacac aaaacaggct caggacttag caagaagtta tggaattcct
     2101 tttattgaaa catcagcaaa gacaagacag ggtgttgatg atgccttcta tacattagtt
     2161 cgagaaattc gaaaacataa agaaaagatg agcaaagatg gtaaaaagaa gaaaaagaag
     2221 tcaaagacaa agtgtgtaat tatgtaagtc gactctagag ggccctattc tatagtgtca
     2281 cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     2341 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2401 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2461 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     2521 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     2581 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2641 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2701 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2761 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2821 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2881 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2941 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3001 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3061 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3121 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3181 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3241 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     3301 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     3361 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     3421 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3481 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3541 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3601 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3661 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3721 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3781 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3841 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3901 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     3961 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4021 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4081 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4141 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4201 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4261 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4321 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4381 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4441 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4501 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     4561 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4621 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4681 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4741 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4801 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4861 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4921 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4981 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5041 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5101 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5161 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5221 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5281 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5341 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5401 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5461 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5521 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5581 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5641 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5701 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5761 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5821 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5881 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5941 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6001 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6061 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6121 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6181 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6241 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6301 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6361 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6421 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6481 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6541 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6601 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6661 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     6721 gatcgggaga tctcccgatc ccctatggtg cactctcagt ac
//
