LOCUS       sequence-422222-        6235 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express constitutively active KRas fused with MBP .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6235)
  AUTHORS   He Y, Faulkner BM, Roberti MA, Bassford DK, Stains C
  TITLE     Standardized Parts for Activation of Small GTPase Signaling in 
            Living Cells.
  JOURNAL   Angew Chem Int Ed Engl. 2024 Jul 26:e202403499. doi: 
            10.1002/anie.202403499.
  PUBMED    39058298
REFERENCE   2  (bases 1 to 6235)
  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"; journalName: "Angew Chem 
            Int Ed Engl. 2024 Jul 26:e202403499. doi: 10.1002/anie.202403499."
COMMENT     Sequence Label: pMAL-MBP-KRas-Q61L
FEATURES             Location/Qualifiers
     source          1..6235
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        19..96
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             97..1179
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(116..135)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1422..1427,1428..1443,1444..1450)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1458..1474
                     /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     1463..1485
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1544..2644
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2618..2641
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2693..2704
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2717..3283
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGLEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     CDS             3308..3325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3337..3423
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3515..3542
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3570..3660
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3661..3729,3730..4521)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3879..3898)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4609..5197
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5098..5117
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5351..5368
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5551..5573
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5567..5758)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 aacgctgccc gaaattccga caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga
       61 tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat
      121 gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc
      181 cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt
      241 cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc
      301 tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat
      361 caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa
      421 gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg
      481 gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt
      541 gatgtctctg accagacacc catcaacagt attattttct cccatgaaga cggtacgcga
      601 ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca
      661 ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat
      721 caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa
      781 accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag
      841 atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatt
      901 tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc
      961 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct
     1021 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc
     1081 accctggcgc ccaatacaca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     1141 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag
     1201 ttagctcact cattaggcac aattctcatg tttgacagct tatcatcgac tgcacggtgc
     1261 accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa
     1321 atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg ttttttgcgc
     1381 cgacatcata acggttctgg caaatattct gaaatgagct gttgacaatt aatcatcggc
     1441 tcgtataatg tgtggaattg tgagcggata acaatttcac acaggaaaca gccagtccgt
     1501 ttaggtgttt tcacgagcaa ttgaccaaca aggaccatag attatgaaaa tcgaagaagg
     1561 taaactggta atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa
     1621 gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata aactggaaga
     1681 gaaattccca caggttgcgg caactggcga tggccctgac attatcttct gggcacacga
     1741 ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt
     1801 ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc
     1861 ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc
     1921 gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag
     1981 cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg
     2041 gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa
     2101 cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa
     2161 tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac
     2221 catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac
     2281 ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg
     2341 tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct
     2401 gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa
     2461 gtcttacgag gaagagttgg tgaaagatcc gcgtattgcc gccactatgg aaaacgccca
     2521 gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac
     2581 tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca
     2641 gactaattcg agctcgaaca acaacaacaa taacaataac aacaacctcg ggatcgaggg
     2701 aaggatttca catatgatga ctgaatataa acttgtggta gttggagctg gtggcgtagg
     2761 caagagtgcc ttgacgatac agctaattca gaatcatttt gtggacgaat atgatccaac
     2821 aatagaggat tcctacagga agcaagtagt aattgatgga gaaacctgtc tcttggatat
     2881 tctcgacaca gcaggtctgg aggagtacag tgcaatgagg gaccagtaca tgaggactgg
     2941 ggagggcttt ctttgtgtat ttgccataaa taatactaaa tcatttgaag atattcacca
     3001 ttatagagaa caaattaaaa gagttaagga ctctgaagat gtacctatgg tcctagtagg
     3061 aaataaatgt gatttgcctt ccagaacagt agacacaaaa caggctcagg acttagcaag
     3121 aagttatgga attcctttta ttgaaacatc agcaaagaca agacagggtg ttgatgatgc
     3181 cttctataca ttagttcgag aaattcgaaa acataaagaa aagatgagca aagatggtaa
     3241 aaagaagaaa aagaagtcaa agacaaagtg tgtaattatg taaggatccg aattccctgc
     3301 aggtaatcat catcatcacc accactaaat aagcttcaaa taaaacgaaa ggctcagtcg
     3361 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     3421 aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga
     3481 cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt
     3541 tttgcgtttc tacaaactct ttcggtccgt tgtttatttt tctaaataca ttcaaatatg
     3601 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3661 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3721 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3781 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3841 gaagaacgtt tcccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3901 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3961 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     4021 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     4081 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     4141 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     4201 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     4261 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     4321 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     4381 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     4441 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4501 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4561 ttccttagga ctgagcgtca accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4621 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4681 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4741 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4801 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4861 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4921 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4981 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5041 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5101 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5161 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5221 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     5281 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     5341 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     5401 ccttacgcat ctgtgcggta tttcacaccg catataaggt gcactgtgac tgggtcatgg
     5461 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     5521 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     5581 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgcagcg
     5641 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     5701 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     5761 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     5821 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     5881 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     5941 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     6001 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     6061 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     6121 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     6181 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggccag gaccc
//
