LOCUS       sequence-422218-        7397 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express split-KRas fragment fused with FKBP and MBP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7397)
  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 7397)
  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: pET21b-KRas/13C-FKBP-MBP
FEATURES             Location/Qualifiers
     source          1..7397
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     441..460
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(679..783)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(809..1264)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(946..967)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1158..1177
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1301..1348)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1344..1362
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1415..1432)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1457..1480)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             complement(2546..2566)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(2573..2893)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     RBS             3480..3502
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3517..3541
                     /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     complement(3541..3560)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3542..3560)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3644..3663)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3869..3946
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3947..5029
                     /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(3966..3985)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5838..6029
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6023..6045)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6131..6270
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6186..6205
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6285..6302)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6456..7044)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6536..6555)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7215..7397,1..609,610..678))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
       61 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
      121 tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
      181 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
      241 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
      301 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
      361 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
      421 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
      481 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
      541 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
      601 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
      661 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
      721 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
      781 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
      841 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
      901 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
      961 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     1021 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     1081 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     1141 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     1201 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     1261 gcgtcccatt cgccaatccg gatatagttc ctcctttcag caaaaaaccc ctcaagaccc
     1321 gtttagaggc cccaaggggt tatgctagtt attgctcagc ggtggcagca gccaactcag
     1381 cttcctttcg ggctttgtta gcagccggat ctcagtggtg gtggtggtgg tgctcgagct
     1441 acgagctgga attagtctgc gcgtctttca gggcttcatc gacagtctga cgaccgctgg
     1501 cggcgttgat caccgcagta cgcacggcat accagaaagc ggacatctgc gggatgttcg
     1561 gcatgatttc acctttctgg gcgttttcca tagtggcggc aatacgtgga tctttcgcca
     1621 actcttcctc gtaagacttc agcgctacgg cacccagcgg tttgtcttta ttaaccgctt
     1681 ccagaccttc atcagtcagc agatagtttt cgaggaactc ttttgccagc tctttgttcg
     1741 gactggcggc gttaatacct gcgctcagca cgccaacgaa cggtttggat ggttgaccct
     1801 tgaaggtcgg cagtaccgtt acaccataat tcactttgct ggtgtcgatg ttggaccatg
     1861 cccacgggcc gttgatggtc atcgctgttt cgcctttatt aaaggcagct tctgcgatgg
     1921 agtaatcggt gtctgcattc atgtgtttgt ttttaatcag gtcaaccagg aaggtcagac
     1981 ccgctttcgc gccagcgtta tccacgccca cgtctttaat gtcgtacttg ccgttttcat
     2041 acttgaacgc ataacccccg tcagcagcaa tcagcggcca ggtgaagtac ggttcttgca
     2101 ggttgaacat cagcgcgctc ttacctttcg ctttcagttc tttatccagc gccgggatct
     2161 cttcccaggt ttttggcggg ttcggcagca ggtctttgtt ataaatcagc gataacgctt
     2221 caacagcgat cgggtaagca atcagcttgc cgttgtaacg tacggcatcc caggtaaacg
     2281 gatacagctt gtcctggaac gctttgtccg gggtgatttc agccaacagg ccagattgag
     2341 cgtagccacc aaagcggtcg tgtgcccaga agataatgtc agggccatcg ccagttgccg
     2401 caacctgtgg gaatttctct tccagtttat ccggatgctc aacggtgact ttaattccgg
     2461 tatctttctc gaatttctta ccgacttcag cgagaccgtt atagccttta tcgccgttaa
     2521 tccagattac cagtttacct tcttcgccct ggaagtacag gttctcaagc ttttccagtt
     2581 ttagaagctc cacatcgaag acgagagtgg catgtggtgg gatgatgcct gggtgcccag
     2641 tggcaccata ggcataatct ggagatatag tcagtttggc tctctgaccc acactcatct
     2701 gggcaacccc ttcttcccag cctcggatca cctcctgctt gcctagcata aacttaaagg
     2761 gcttgtttct gtcccgggag gaatcaaatt tctttccatc ttcaagcatc ccggtgtagt
     2821 gcaccacgca ggtctggccg cgcttgggga aggtgcgccc gtctcctggg gagatggttt
     2881 ccacctgcac tccgccccgg gacgcgtacg agatctgacc tccgccagat gagccacctc
     2941 cctgcaggcg cgccgtgtac acgagctccc aattgggatc cccaccatct ttgctcatct
     3001 tttctttatg ttttcgaatt tctcgaacta atgtatagaa ggcatcatca acaccctgtc
     3061 ttgtctttgc tgatgtttca ataaaaggaa ttccataact tcttgctaag tcctgagcct
     3121 gttttgtgtc tactgttctg gaaggcaaat cacatttatt tcctactagg accataggta
     3181 catcttcaga gtccttaact cttttaattt gttctctata atggtgaata tcttcaaatg
     3241 atttagtatt atttatggca aatacacaaa gaaagccctc cccagtcctc atgtactggt
     3301 ccctcattgc actgtactcc tccagacctg ctgtgtcgag aatatccaag agacaggttt
     3361 ctccatcaat tactacttgc ttcctgtagg aatcctctat tgttggatca tattcgtcca
     3421 caaaatgatt ctgaattagc tgtatcgtca aggcactctt gcctacgccc atatgtatat
     3481 ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc
     3541 ccctatagtg agtcgtatta atttcgcggg atcgagatct cgatcctcta cgccggacgc
     3601 atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc
     3661 accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt
     3721 atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc
     3781 cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag
     3841 tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg
     3901 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
     3961 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     4021 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     4081 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     4141 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     4201 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     4261 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     4321 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     4381 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
     4441 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     4501 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     4561 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     4621 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     4681 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     4741 tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
     4801 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     4861 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
     4921 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4981 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     5041 ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct
     5101 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
     5161 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg
     5221 gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa
     5281 tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga
     5341 agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt
     5401 tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac
     5461 gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa
     5521 tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca
     5581 ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg
     5641 tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata
     5701 ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc
     5761 gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc
     5821 cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta
     5881 tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg
     5941 cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc
     6001 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     6061 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     6121 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
     6181 ctatgcggca tcagagcaga ttgtactgag agtgcaccat tgcggtgtga aataccgcac
     6241 agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg
     6301 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     6361 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     6421 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     6481 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     6541 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     6601 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     6661 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6721 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6781 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6841 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     6901 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6961 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     7021 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     7081 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     7141 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     7201 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     7261 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     7321 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     7381 ttatcagcaa taaacca
//
