LOCUS       sequence-422238-        6220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express constitutively active 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-Q61L
FEATURES             Location/Qualifiers
     source          1..6220
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(6..12,13..28,29..34))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(277..1359)
                     /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     1321..1340
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1360..1437)
                     /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             1933..2124
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2118..2140)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2323..2340)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2494..3082)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2574..2593)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3170..3961,3962..4030))
                     /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     3793..3812
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4031..4121)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(4149..4176)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4268..4354)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4366..4383)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4972..4983)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(5032..6132)
                     /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     complement(5035..5058)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     primer_bind     complement(6191..6213)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6202..6218
                     /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)."
ORIGIN
        1 ccacacatta tacgagccga tgattaattg tcaacagctc atttcagaat atttgccaga
       61 accgttatga tgtcggcgca aaaaacatta tccagaacgg gagtgcgcct tgagcgacac
      121 gaattatgca gtgatttacg acctgcacag ccataccaca gcttccgatg gctgcctgac
      181 gccagaagca ttggtgcacc gtgcagtcga tgataagctg tcaaacatga gaattgtgcc
      241 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
      301 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgtgt
      361 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
      421 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
      481 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
      541 gtatcccact accgaaatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
      601 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
      661 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
      721 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
      781 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
      841 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
      901 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
      961 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     1021 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     1081 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     1141 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     1201 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     1261 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     1321 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     1381 ctcttccggg cgctatcatg ccataccgcg aaaggttttg caccattcga tggtgtcgga
     1441 atttcgggca gcgttgggtc ctggccacgg gtgcgcatga tcgtgctcct gtcgttgagg
     1501 acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag
     1561 cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc
     1621 ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg
     1681 ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta
     1741 acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc
     1801 agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc
     1861 gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac
     1921 acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga
     1981 agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac
     2041 atctgtgaat cgctgcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc
     2101 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     2161 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     2221 cggggcgcag ccatgaccca gtcacagtgc accttatatg cggtgtgaaa taccgcacag
     2281 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct
     2341 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2401 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2461 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2521 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2581 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2641 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2701 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2761 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2821 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2881 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     2941 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3001 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3061 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt tgacgctcag
     3121 tcctaaggaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3181 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     3241 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3301 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3361 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3421 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3481 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3541 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     3601 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     3661 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     3721 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     3781 cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggga
     3841 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     3901 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     3961 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     4021 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     4081 tgagcggata catatttgaa tgtatttaga aaaataaaca acggaccgaa agagtttgta
     4141 gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt aatttgatgc ctggcagttt
     4201 atggcgggcg tcctgcccgc caccctccgg gccgttgctt cgcaacgttc aaatccgctc
     4261 ccggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata aaacgaaagg
     4321 cccagtcttt cgactgagcc tttcgtttta tttgaagctt atttagtggt ggtgatgatg
     4381 atgattacct gcagggaatt cggatcctta gggttgagtt tccggaggct cgagggcagc
     4441 taggatagcc tcatcaaaca cattcttcag acctctctgt gtaagtgcag aacactccac
     4501 atacttgaca gccttcaggt cacgggccag cttttcagca gtctctggag tgataggctt
     4561 ctgtttgttc ttggcaagtt tctcaatagt agaggggtca tctctgagat caatttgagt
     4621 cccaacaagc aagaaaggag tctttggaca gtggtgagtt atctcaggca cccacttttc
     4681 tttcacgttt tcaaatgaag atggagagac cactgaaaaa cagactagaa atacatctgt
     4741 ttgtggataa ctcagcggtc gtaatctgtc ataatcctct agccctgcag tatcaaaaag
     4801 tccaagagta tatggttctc caccaatcat aactgtgact gcatagttgt caaaaacagt
     4861 cggtacatac tccgatggaa atttgtttgt tgtgtaggat atcaggagac atgttttacc
     4921 aacagcacca tcgcccacaa caacacactt aattgtctgc atatgtgaaa tccttccctc
     4981 gatcccgagg ttgttgttat tgttattgtt gttgttgttc gagctcgaat tagtctgcgc
     5041 gtctttcagg gcttcatcga cagtctgacg accgctggcg gcgttgatca ccgcagtacg
     5101 cacggcatac cagaaagcgg acatctgcgg gatgttcggc atgatttcac ctttctgggc
     5161 gttttccata gtggcggcaa tacgcggatc tttcaccaac tcttcctcgt aagacttcag
     5221 cgctacggca cccagcggtt tgtctttatt aaccgcttcc agaccttcat cagtcagcag
     5281 atagttttcg aggaactctt ttgccagctc tttgttcgga ctggcggcgt taatacctgc
     5341 gctcagcacg ccaacgaacg gtttggatgg ttgacccttg aaggtcggca gtaccgttac
     5401 accataattc actttgctgg tgtcgatgtt ggaccatgcc cacgggccgt tgatggtcat
     5461 cgctgtttcg cctttattaa aggcagcttc tgcgatggag taatcggtgt ctgcattcat
     5521 gtgtttgttt ttaatcaggt caaccaggaa ggtcagaccc gctttcgcgc cagcgttatc
     5581 cacgcccacg tctttaatgt cgtacttgcc gttttcatac ttgaacgcat aacccccgtc
     5641 agcagcaatc agcggccagg tgaagtacgg ttcttgcagg ttgaacatca gcgcgctctt
     5701 acctttcgct ttcagttctt tatccagcgc cgggatctct tcccaggttt ttggcgggtt
     5761 cggcagcaga tctttgttat aaatcagcga taacgcttca acagcgatcg ggtaagcaat
     5821 cagcttgccg ttgtaacgta cggcatccca ggtaaacgga tacagcttgt cctggaacgc
     5881 tttgtccggg gtgatttcag ccaacaggcc agattgagcg tagccaccaa agcggtcgtg
     5941 tgcccagaag ataatgtcag ggccatcgcc agttgccgca acctgtggga atttctcttc
     6001 cagtttatcc ggatgctcaa cggtgacttt aattccggta tctttctcga atttcttacc
     6061 gacttcagcg agaccgttat agcctttatc gccgttaatc cagattacca gtttaccttc
     6121 ttcgattttc ataatctatg gtccttgttg gtcaattgct cgtgaaaaca cctaaacgga
     6181 ctggctgttt cctgtgtgaa attgttatcc gctcacaatt
//
