LOCUS       sequence-217480-        6762 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses YFP-KRAS4B fusion protein used for FRET studies in 
            eukaryotic 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-YFP-KRAS4B
FEATURES             Location/Qualifiers
     source          1..6762
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        25..129
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(130..198,199..990)
                     /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     complement(348..367)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1161..1749
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1650..1669
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1903..1920
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2037..2058
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2073..2078,2079..2096,2097..2103)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2111..2127
                     /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)."
     primer_bind     2116..2138
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2135..2151
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2135..2151
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2200..2321
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2211..2230)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2265..2284
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2495..3289)
                     /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(2606..2625)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3216..3235
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3356..3685)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3370..3505
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3424..3443)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3670..3690
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3699..4127)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3809..3830)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4021..4040
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    4173..4397
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4386..4403
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        4423..4441
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4423..4440
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(4463..5029)
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     CDS             complement(join(5063..5773,5774..5776,5777..5779))
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(5063..5773,5774..5776,5777..5779))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5105..5126)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     5454..5473
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5713..5734
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5874..5893)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5875..5893)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5938..6141)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5967..5987)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6142..6521
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6693..6712
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
       61 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
      121 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
      181 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
      241 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
      301 tttcgccccg aagaacgttt tccaatgatg agcacattta aagttctgct atgtggcgcg
      361 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
      421 aatgacttgg ttgagtactc acctgtcaca gaaaagcatc ttacggatgg catgacagta
      481 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
      541 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
      601 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
      661 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
      721 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
      781 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
      841 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
      901 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
      961 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     1021 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     1081 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     1141 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     1201 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     1261 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     1321 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     1381 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     1441 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     1501 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     1561 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     1621 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     1681 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     1741 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     1801 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     1861 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     1921 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     1981 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     2041 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     2101 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     2161 gccaagctct agctagaggt cgacggtata cagacatgat aagatacatt gatgagtttg
     2221 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     2281 ttgctttatt tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc
     2341 agcatgagat ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag
     2401 cccaaccttt catagaaggc ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc
     2461 gcttggtcgg tcatttcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat
     2521 agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag
     2581 cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc
     2641 ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca
     2701 tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc
     2761 gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat
     2821 catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg
     2881 cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag
     2941 ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca
     3001 cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc
     3061 aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca
     3121 gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga
     3181 acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct
     3241 ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc
     3301 ctcatcctgt ctcttgatca gatccgaaaa tggatataca agctcccggg agctttttgc
     3361 aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca gaggcagagg
     3421 cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg
     3481 aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact
     3541 aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc
     3601 tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg
     3661 actttccaca ccctaactga cacacattcc acagaattaa ttcgcgttaa atttttgtta
     3721 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     3781 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     3841 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     3901 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     3961 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     4021 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     4081 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtggg gataccccct
     4141 agagccccag ctggttcttt ccgcctcaga agccatagag cccaccgcat ccccagcatg
     4201 cctgctattg tcttcccaat cctccccctt gctgtcctgc cccaccccac cccccagaat
     4261 agaatgacac ctactcagac aatgcgatgc aatttcctca ttttattagg aaaggacagt
     4321 gggagtggca ccttccaggg tcaaggaagg cacgggggag gggcaaacaa cagatggctg
     4381 gcaactagaa ggcacagtcg aggctgatca gcgagctcta gcatttaggt gacactatag
     4441 aatagggccc tctagagtcg acttacataa ttacacactt tgtctttgac ttctttttct
     4501 tctttttacc atctttgctc atcttttctt tatgttttcg aatttctcga actaatgtat
     4561 agaaggcatc atcaacaccc tgtcttgtct ttgctgatgt ttcaataaaa ggaattccat
     4621 aacttcttgc taagtcctga gcctgttttg tgtctactgt tctagaaggc aaatcacatt
     4681 tatttcctac taggaccata ggtacatctt cagagtcctt aactctttta atttgttctc
     4741 tataatggtg aatatcttca aatgatttag tattatttat ggcaaataca caaagaaagc
     4801 cctccccagt cctcatgtac tggtccctca ttgcactgta ctcctcttga cctgctgtgt
     4861 cgagaatatc caagagacag gtttctccat caattactac ttgcttcctg taggaatcct
     4921 ctattgttgg atcatattcg tccacaaaat gattctgaat tagctgtatc gtcaaggcac
     4981 tcttgcctac gccaccagct ccaactacca caagtttata ttcagtcatg gtggcggcgc
     5041 tagccagctt ggatccggcg cccttgtaca gctcgtccat gccgagagtg atcccggcgg
     5101 cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct ttgctcaggg
     5161 cggactggta gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg
     5221 tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg tggcggatct
     5281 tgaagttcac cttgatgccg ttcttctgct tgtcggccat gatatagacg ttgtggctgt
     5341 tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag tcgatgccct
     5401 tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt
     5461 agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg ggcatggcgg
     5521 acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg ggcgaagcac ttcaggccgt
     5581 agccgaaggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg gtgcagatga
     5641 acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac acgctgaact
     5701 tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg gtgaacagct
     5761 cctcgccctt gctcaccatc tcgagcggcc gccagtgtga tggatatctg cagaattcca
     5821 gcacactggc ggccgttact agtggatccg agctcggtac caagcttggg tctccctata
     5881 gtgagtcgta ttaatttcga taagccagta agcagtgggt tctctagtta gccagagagc
     5941 tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca atggggcgga
     6001 gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac tcccattgac
     6061 gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac gcccattgat
     6121 gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga tgtactgcca
     6181 agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc atttaccgtc
     6241 attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg ccaagtgggc
     6301 agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg gcgttactat
     6361 gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg
     6421 gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa ctaatgaccc
     6481 cgtaattgat tactattaat aactagtcaa taatcaatgt caacgcgtat atctggcccg
     6541 tacatcgcga agcagcgcaa aacgcctaac cctaagcaga ttcttcatgc aattgtcggt
     6601 caagccttgc cttgttgtag cttaaatttt gctcgcgcac tactcagcga cctccaacac
     6661 acaagcaggg agcagatact ggcttaacta tgcggcatca gagcagattg tactgagagt
     6721 gcaccatagg ggatcgggag atctcccgat ccgtcgacgt ca
//
