LOCUS       sequence-422237-        6220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express dominant negative Cdc42 fused with MBP .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6220)
  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 6220)
  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-Cdc42-T17N
FEATURES             Location/Qualifiers
     source          1..6220
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    21..37
                     /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     26..48
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             107..1207
                     /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     1181..1204
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1256..1267
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1856..1873
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1885..1971
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2063..2090
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2118..2208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2209..2277,2278..3069)
                     /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(2427..2446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3157..3745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3646..3665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3899..3916
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4099..4121
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4115..4306)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        4802..4879
                     /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             4880..5962
                     /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(4899..4918)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6205..6210,6211..6220,1..6,7..13)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
ORIGIN
        1 ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagccagtc
       61 cgtttaggtg ttttcacgag caattgacca acaaggacca tagattatga aaatcgaaga
      121 aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg
      181 taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga
      241 agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca
      301 cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc
      361 gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat
      421 tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa
      481 cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa
      541 gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga
      601 cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga
      661 taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat
      721 gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat
      781 gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt
      841 aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc
      901 aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg aaaactatct
      961 gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct
     1021 gaagtcttac gaggaagagt tggtgaaaga tccgcgtatt gccgccacta tggaaaacgc
     1081 ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg
     1141 tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc
     1201 gcagactaat tcgagctcga acaacaacaa caataacaat aacaacaacc tcgggatcga
     1261 gggaaggatt tcacatatgc agacaattaa gtgtgttgtt gtgggcgatg gcgccgttgg
     1321 taaaaactgt ctcctgatat cctacacaac aaacaaattt ccatcggagt atgtaccgac
     1381 tgtttttgac aactatgcag tcacagttat gattggtgga gaaccatata ctcttggact
     1441 ttttgatact gcagggcaag aggattatga cagattacga ccgctgagtt atccacaaac
     1501 agatgtattt ctagtctgtt tttcagtggt ctctccatct tcatttgaaa acgtgaaaga
     1561 aaagtgggtg cctgagataa ctcaccactg tccaaagact cctttcttgc ttgttgggac
     1621 tcaaattgat ctcagagatg acccctctac tattgagaaa cttgccaaga acaaacagaa
     1681 gcctatcact ccagagactg ctgaaaagct ggcccgtgac ctgaaggctg tcaagtatgt
     1741 ggagtgttct gcacttacac agagaggtct gaagaatgtg tttgatgagg ctatcctagc
     1801 tgccctcgag cctccggaaa ctcaacccta aggatccgaa ttccctgcag gtaatcatca
     1861 tcatcaccac cactaaataa gcttcaaata aaacgaaagg ctcagtcgaa agactgggcc
     1921 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga
     1981 gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa
     2041 actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta
     2101 caaactcttt cggtccgttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2161 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2221 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2281 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     2341 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttc
     2401 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc
     2461 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     2521 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     2581 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     2641 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     2701 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     2761 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     2821 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     2881 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     2941 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     3001 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     3061 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt ccttaggact
     3121 gagcgtcaac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3181 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3241 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3301 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3361 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3421 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3481 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3541 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3601 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3661 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3721 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3781 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3841 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3901 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3961 gtgcggtatt tcacaccgca tataaggtgc actgtgactg ggtcatggct gcgccccgac
     4021 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4081 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4141 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgcagcgat tcacagatgt
     4201 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     4261 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     4321 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     4381 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     4441 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     4501 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     4561 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     4621 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     4681 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     4741 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgaaattc
     4801 cgacaccatc gaatggtgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     4861 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     4921 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     4981 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     5041 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     5101 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     5161 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     5221 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     5281 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     5341 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     5401 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     5461 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     5521 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     5581 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     5641 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atttcggtag tgggatacga
     5701 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     5761 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     5821 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     5881 acaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     5941 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     6001 cacaattctc atgtttgaca gcttatcatc gactgcacgg tgcaccaatg cttctggcgt
     6061 caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg cataattcgt
     6121 gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc
     6181 tggcaaatat tctgaaatga gctgttgaca attaatcatc
//
