LOCUS       sequence-422221-        6235 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express dominant negative 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-S17N
FEATURES             Location/Qualifiers
     source          1..6235
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    14..30
                     /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     19..41
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             100..1200
                     /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     1174..1197
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1249..1260
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1273..1839
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGAGGVGKNALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     CDS             1864..1881
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1893..1979
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2071..2098
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2126..2216
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2217..2285,2286..3077)
                     /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(2435..2454)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3165..3753
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3654..3673
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3907..3924
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4107..4129
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4123..4314)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        4810..4887
                     /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             4888..5970
                     /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(4907..4926)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6213..6218,6219..6234,6235,1..6)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
ORIGIN
        1 ataatgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcca gtccgtttag
       61 gtgttttcac gagcaattga ccaacaagga ccatagatta tgaaaatcga agaaggtaaa
      121 ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt cggtaagaaa
      181 ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa
      241 ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc
      301 tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag
      361 gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac
      421 ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca
      481 aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg
      541 ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt
      601 tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct
      661 ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca
      721 gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc
      781 aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta
      841 ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt
      901 aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact
      961 gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct
     1021 tacgaggaag agttggtgaa agatccgcgt attgccgcca ctatggaaaa cgcccagaaa
     1081 ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg
     1141 gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact
     1201 aattcgagct cgaacaacaa caacaataac aataacaaca acctcgggat cgagggaagg
     1261 atttcacata tgatgactga atataaactt gtggtagttg gagctggtgg cgtaggcaag
     1321 aatgccttga cgatacagct aattcagaat cattttgtgg acgaatatga tccaacaata
     1381 gaggattcct acaggaagca agtagtaatt gatggagaaa cctgtctctt ggatattctc
     1441 gacacagcag gtcaagagga gtacagtgca atgagggacc agtacatgag gactggggag
     1501 ggctttcttt gtgtatttgc cataaataat actaaatcat ttgaagatat tcaccattat
     1561 agagaacaaa ttaaaagagt taaggactct gaagatgtac ctatggtcct agtaggaaat
     1621 aaatgtgatt tgccttctag aacagtagac acaaaacagg ctcaggactt agcaagaagt
     1681 tatggaattc cttttattga aacatcagca aagacaagac agggtgttga tgatgccttc
     1741 tatacattag ttcgagaaat tcgaaaacat aaagaaaaga tgagcaaaga tggtaaaaag
     1801 aagaaaaaga agtcaaagac aaagtgtgta attatgtaag gatccgaatt ccctgcaggt
     1861 aatcatcatc atcaccacca ctaaataagc ttcaaataaa acgaaaggct cagtcgaaag
     1921 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc
     1981 cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc
     2041 cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg
     2101 cgtttctaca aactctttcg gtccgttgtt tatttttcta aatacattca aatatgtatc
     2161 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     2221 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     2281 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     2341 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     2401 aacgtttccc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg
     2461 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     2521 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     2581 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     2641 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     2701 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     2761 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     2821 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     2881 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     2941 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     3001 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     3061 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttcc
     3121 ttaggactga gcgtcaaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3181 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3241 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3301 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3361 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3421 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3481 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3541 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3601 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3661 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3721 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3781 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3841 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3901 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     3961 acgcatctgt gcggtatttc acaccgcata taaggtgcac tgtgactggg tcatggctgc
     4021 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4081 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4141 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gcagcgattc
     4201 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     4261 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     4321 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     4381 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     4441 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     4501 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     4561 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     4621 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     4681 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     4741 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggccaggacc caacgctgcc
     4801 cgaaattccg acaccatcga atggtgcaaa acctttcgcg gtatggcatg atagcgcccg
     4861 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     4921 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     4981 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     5041 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     5101 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     5161 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     5221 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     5281 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     5341 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     5401 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     5461 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     5521 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     5581 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     5641 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ttcggtagtg
     5701 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     5761 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     5821 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     5881 cccaatacac aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     5941 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
     6001 tcattaggca caattctcat gtttgacagc ttatcatcga ctgcacggtg caccaatgct
     6061 tctggcgtca ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta aatcactgca
     6121 taattcgtgt cgctcaaggc gcactcccgt tctggataat gttttttgcg ccgacatcat
     6181 aacggttctg gcaaatattc tgaaatgagc tgttgacaat taatcatcgg ctcgt
//
