LOCUS       sequence-212810-        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"
     promoter        32..136
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(137..205,206..997)
                     /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(355..374)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1168..1756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1657..1676
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1910..1927
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2044..2065
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2080..2085,2086..2103,2104..2110)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2118..2134
                     /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     2123..2145
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2142..2158
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2142..2158
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2207..2328
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2218..2237)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2272..2291
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2502..3296)
                     /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(2613..2632)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3223..3242
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3363..3692)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3377..3512
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3431..3450)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3677..3697
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3706..4134)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3816..3837)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4028..4047
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    4180..4404
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4393..4410
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        4430..4448
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4430..4447
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(4470..5036)
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     CDS             complement(join(5070..5780,5781..5783,5784..5786))
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5112..5133)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     5461..5480
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5720..5741
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5881..5900)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5882..5900)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5945..6148)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5974..5994)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6149..6528
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6700..6719
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
       61 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
      121 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
      181 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
      241 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
      301 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
      361 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
      421 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
      481 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
      541 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
      601 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
      661 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
      721 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
      781 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
      841 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
      901 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
      961 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     1021 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     1081 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     1141 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     1201 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     1261 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     1321 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     1381 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     1441 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     1501 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     1561 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     1621 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     1681 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     1741 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     1801 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     1861 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     1921 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     1981 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     2041 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     2101 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     2161 tgattacgcc aagctctagc tagaggtcga cggtatacag acatgataag atacattgat
     2221 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     2281 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgg ggtgggcgaa
     2341 gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat
     2401 tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt
     2461 gggcgtcgct tggtcggtca tttcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga
     2521 aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag
     2581 cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc
     2641 tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt
     2701 tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg
     2761 ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg
     2821 tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga
     2881 tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt
     2941 gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc
     3001 cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca
     3061 gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt
     3121 tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac
     3181 agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat
     3241 agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatgcga
     3301 aacgatcctc atcctgtctc ttgatcagat ccgaaaatgg atatacaagc tcccgggagc
     3361 tttttgcaaa agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag
     3421 gcagaggcgg cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa
     3481 tgggcggaac tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt
     3541 gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt
     3601 ccacacctgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag
     3661 cctggggact ttccacaccc taactgacac acattccaca gaattaattc gcgttaaatt
     3721 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     3781 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     3841 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     3901 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     3961 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     4021 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     4081 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtggggat
     4141 accccctaga gccccagctg gttctttccg cctcagaagc catagagccc accgcatccc
     4201 cagcatgcct gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc
     4261 ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa
     4321 ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag
     4381 atggctggca actagaaggc acagtcgagg ctgatcagcg agctctagca tttaggtgac
     4441 actatagaat agggccctct agagtcgact tacataatta cacactttgt ctttgacttc
     4501 tttttcttct ttttaccatc tttgctcatc ttttctttat gttttcgaat ttctcgaact
     4561 aatgtataga aggcatcatc aacaccctgt cttgtctttg ctgatgtttc aataaaagga
     4621 attccataac ttcttgctaa gtcctgagcc tgttttgtgt ctactgttct agaaggcaaa
     4681 tcacatttat ttcctactag gaccataggt acatcttcag agtccttaac tcttttaatt
     4741 tgttctctat aatggtgaat atcttcaaat gatttagtat tatttatggc aaatacacaa
     4801 agaaagccct ccccagtcct catgtactgg tccctcattg cactgtactc ctcttgacct
     4861 gctgtgtcga gaatatccaa gagacaggtt tctccatcaa ttactacttg cttcctgtag
     4921 gaatcctcta ttgttggatc atattcgtcc acaaaatgat tctgaattag ctgtatcgtc
     4981 aaggcactct tgcctacgcc accagctcca actaccacaa gtttatattc agtcatggtg
     5041 gcggcgctag ccagcttgga tccggcgccc ttgtacagct cgtccatgcc gagagtgatc
     5101 ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg
     5161 ctcagggcgg actgggtgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg
     5221 atgggggtgt tctgctggta gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg
     5281 cggatcttga agtgggcctt gatgccgttc ttctgcttgt cggcggtgat atagacgttg
     5341 tggctgatgt agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg
     5401 atgcccttca gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg
     5461 gtcttgtagt tgccgtcgtc cttgaagaag atggtacgct cctggacgta gccttcgggc
     5521 atggcggact tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc
     5581 acgccccagg tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg
     5641 cagatgaact tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg
     5701 ctgaacctgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg
     5761 aacagctcct cgcccttgct caccatctcg agcggccgcc agtgtgatgg atatctgcag
     5821 aattccagca cactggcggc cgttactagt ggatccgagc tcggtaccaa gcttgggtct
     5881 ccctatagtg agtcgtatta atttcgataa gccagtaagc agtgggttct ctagttagcc
     5941 agagagctct gcttatatag acctcccacc gtacacgcct accgcccatt tgcgtcaatg
     6001 gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa aacaaactcc
     6061 cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc tatccacgcc
     6121 cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat acgtagatgt
     6181 actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt
     6241 taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca
     6301 agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg
     6361 ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc
     6421 caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta
     6481 atgaccccgt aattgattac tattaataac tagtcaataa tcaatgtcaa cgcgtatatc
     6541 tggcccgtac atcgcgaagc agcgcaaaac gcctaaccct aagcagattc ttcatgcaat
     6601 tgtcggtcaa gccttgcctt gttgtagctt aaattttgct cgcgcactac tcagcgacct
     6661 ccaacacaca agcagggagc agatactggc ttaactatgc ggcatcagag cagattgtac
     6721 tgagagtgca ccatagggga tcgggagatc tcccgatccg tc
//
