LOCUS       sequence-422220-        6028 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express split-KRas fragment fused with FRB and MBP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6028)
  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 6028)
  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-FRB-KRas/N12
FEATURES             Location/Qualifiers
     source          1..6028
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(792..797,798..813,814..820)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    828..844
                     /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     833..855
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             914..2014
                     /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     1988..2011
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2063..2074
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2087..2356
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     CDS             2471..2488
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2500..2586
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2678..2705
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2733..2823
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2824..2892,2893..3684)
                     /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(3042..3061)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3772..4360
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4261..4280
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4514..4531
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4714..4736
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4730..4921)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        5417..5494
                     /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             join(5495..6028,1..549)
                     /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(5514..5533)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg
       61 gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc
      121 gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt
      181 cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc
      241 gagtccgggc tgcgcgttgg tgcggatatt tcggtagtgg gatacgacga taccgaagac
      301 agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa
      361 accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg
      421 ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacaca aaccgcctct
      481 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
      541 gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac aattctcatg
      601 tttgacagct tatcatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg
      661 gaagctgtgg tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg
      721 cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct
      781 gaaatgagct gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata
      841 acaatttcac acaggaaaca gccagtccgt ttaggtgttt tcacgagcaa ttgaccaaca
      901 aggaccatag attatgaaaa tcgaagaagg taaactggta atctggatta acggcgataa
      961 aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt
     1021 caccgttgag catccggata aactggaaga gaaattccca caggttgcgg caactggcga
     1081 tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct
     1141 gttggctgaa atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga
     1201 tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct
     1261 gatttataac aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct
     1321 ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta
     1381 cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa
     1441 gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct
     1501 ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc
     1561 tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat
     1621 cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc
     1681 caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct
     1741 ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa
     1801 agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg tgaaagatcc
     1861 gcgtattgcc gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca
     1921 gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca
     1981 gactgtcgat gaagccctga aagacgcgca gactaattcg agctcgaaca acaacaacaa
     2041 taacaataac aacaacctcg ggatcgaggg aaggatttca catatggaga tgtggcatga
     2101 aggcctggaa gaggcatctc gtttgtactt tggggaaagg aacgtgaaag gcatgtttga
     2161 ggtgctggag cccttgcatg ctatgatgga acggggcccc cagactctga aggaaacatc
     2221 ctttaatcag gcctatggtc gagatttaat ggaggcccaa gagtggtgca ggaagtacat
     2281 gaaatcaggg aatgtcaagg acctcaccca agcctgggac ctctattatc atgtgttccg
     2341 acgaatctca aagcagcaga tctcgtacgc gtcccggggc ggtggctcat ctggcggagg
     2401 tgagctcatg actgaatata aacttgtggt agttggagct ggttaaggat ccgaattccc
     2461 tgcaggtaat catcatcatc accaccacta aataagcttc aaataaaacg aaaggctcag
     2521 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     2581 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
     2641 ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc
     2701 ctttttgcgt ttctacaaac tctttcggtc cgttgtttat ttttctaaat acattcaaat
     2761 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2821 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2881 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     2941 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     3001 cccgaagaac gtttcccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     3061 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     3121 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     3181 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     3241 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     3301 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     3361 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3421 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3481 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     3541 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     3601 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3661 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     3721 gatttcctta ggactgagcg tcaaccccgt agaaaagatc aaaggatctt cttgagatcc
     3781 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3841 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3901 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3961 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4021 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4081 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4141 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4201 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4261 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4321 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4381 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4441 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4501 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     4561 tctccttacg catctgtgcg gtatttcaca ccgcatataa ggtgcactgt gactgggtca
     4621 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4681 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4741 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgca
     4801 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     4861 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     4921 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     4981 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     5041 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     5101 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     5161 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     5221 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     5281 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     5341 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa
     5401 cgctgcccga aattccgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata
     5461 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     5521 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     5581 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     5641 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     5701 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     5761 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     5821 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     5881 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     5941 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     6001 gggcgtggag catctggtcg cattgggt
//
