LOCUS       sequence-422236-        6028 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express split-Cdc42 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-Cdc42/N12
FEATURES             Location/Qualifiers
     source          1..6028
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(81..86,87..102,103..109)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    117..133
                     /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     122..144
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             203..1303
                     /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     1277..1300
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1352..1363
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1376..1645
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     CDS             1760..1777
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1789..1875
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1967..1994
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2022..2112
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2113..2181,2182..2973)
                     /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(2331..2350)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3061..3649
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3550..3569
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3803..3820
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4003..4025
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4019..4210)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        4706..4783
                     /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             4784..5866
                     /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(4803..4822)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa cggttctggc
       61 aaatattctg aaatgagctg ttgacaatta atcatcggct cgtataatgt gtggaattgt
      121 gagcggataa caatttcaca caggaaacag ccagtccgtt taggtgtttt cacgagcaat
      181 tgaccaacaa ggaccataga ttatgaaaat cgaagaaggt aaactggtaa tctggattaa
      241 cggcgataaa ggctataacg gtctcgctga agtcggtaag aaattcgaga aagataccgg
      301 aattaaagtc accgttgagc atccggataa actggaagag aaattcccac aggttgcggc
      361 aactggcgat ggccctgaca ttatcttctg ggcacacgac cgctttggtg gctacgctca
      421 atctggcctg ttggctgaaa tcaccccgga caaagcgttc caggacaagc tgtatccgtt
      481 tacctgggat gccgtacgtt acaacggcaa gctgattgct tacccgatcg ctgttgaagc
      541 gttatcgctg atttataaca aagatctgct gccgaacccg ccaaaaacct gggaagagat
      601 cccggcgctg gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt tcaacctgca
      661 agaaccgtac ttcacctggc cgctgattgc tgctgacggg ggttatgcgt tcaagtatga
      721 aaacggcaag tacgacatta aagacgtggg cgtggataac gctggcgcga aagcgggtct
      781 gaccttcctg gttgacctga ttaaaaacaa acacatgaat gcagacaccg attactccat
      841 cgcagaagct gcctttaata aaggcgaaac agcgatgacc atcaacggcc cgtgggcatg
      901 gtccaacatc gacaccagca aagtgaatta tggtgtaacg gtactgccga ccttcaaggg
      961 tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg ccagtccgaa
     1021 caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag gtctggaagc
     1081 ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg aagagttggt
     1141 gaaagatccg cgtattgccg ccactatgga aaacgcccag aaaggtgaaa tcatgccgaa
     1201 catcccgcag atgtccgctt tctggtatgc cgtgcgtact gcggtgatca acgccgccag
     1261 cggtcgtcag actgtcgatg aagccctgaa agacgcgcag actaattcga gctcgaacaa
     1321 caacaacaat aacaataaca acaacctcgg gatcgaggga aggatttcac atatggagat
     1381 gtggcatgaa ggcctggaag aggcatctcg tttgtacttt ggggaaagga acgtgaaagg
     1441 catgtttgag gtgctggagc ccttgcatgc tatgatggaa cggggccccc agactctgaa
     1501 ggaaacatcc tttaatcagg cctatggtcg agatttaatg gaggcccaag agtggtgcag
     1561 gaagtacatg aaatcaggga atgtcaagga cctcacccaa gcctgggacc tctattatca
     1621 tgtgttccga cgaatctcaa agcagcagat ctcgtacgcg tcccggggcg gtggctcatc
     1681 tggcggaggt gagctccaga caattaagtg tgttgttgtg ggcgatggtg cttaaggatc
     1741 cgaattccct gcaggtaatc atcatcatca ccaccactaa ataagcttca aataaaacga
     1801 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     1861 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg
     1921 tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg
     1981 acggatggcc tttttgcgtt tctacaaact ctttcggtcc gttgtttatt tttctaaata
     2041 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     2101 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     2161 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     2221 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     2281 agttttcgcc ccgaagaacg tttcccaatg atgagcactt ttaaagttct gctatgtggc
     2341 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     2401 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     2461 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     2521 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     2581 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     2641 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     2701 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     2761 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     2821 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     2881 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     2941 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     3001 ctttagattg atttccttag gactgagcgt caaccccgta gaaaagatca aaggatcttc
     3061 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3121 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3181 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3241 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3301 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3361 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3421 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3481 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3541 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3601 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3661 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3721 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3781 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     3841 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatataag gtgcactgtg
     3901 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3961 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4021 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     4081 ggtcgtgcag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     4141 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     4201 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     4261 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     4321 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     4381 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     4441 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     4501 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     4561 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     4621 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc
     4681 aggacccaac gctgcccgaa attccgacac catcgaatgg tgcaaaacct ttcgcggtat
     4741 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     4801 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     4861 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     4921 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     4981 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     5041 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     5101 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     5161 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     5221 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     5281 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     5341 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     5401 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     5461 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
     5521 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
     5581 ggatatttcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
     5641 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
     5701 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
     5761 aaaaaccacc ctggcgccca atacacaaac cgcctctccc cgcgcgttgg ccgattcatt
     5821 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     5881 atgtaagtta gctcactcat taggcacaat tctcatgttt gacagcttat catcgactgc
     5941 acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag
     6001 gtcgtaaatc actgcataat tcgtgtcg
//
