LOCUS       sequence-422235-        7427 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express split-Cdc42 fragment fused with FKBP and MBP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7427)
  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 7427)
  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-Cdc42/13C-FKBP-MBP
FEATURES             Location/Qualifiers
     source          1..7427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      371..959
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     860..879
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1113..1130
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1145..1284
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1210..1229)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1370..1392
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1386..1577)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2386..3468)
                     /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     3430..3449
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3469..3546)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3752..3771
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3855..3874
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3855..3873
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3874..3898
                     /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)."
     RBS             3913..3935
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4552..4872
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             4879..4899
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     5965..5988
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             6013..6030
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6083..6101)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6097..6144
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6181..6636
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6268..6287)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6478..6499
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6662..6766
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6767..6835,6836..7427,1..200)
                     /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"
     primer_bind     complement(6985..7004)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
       61 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
      121 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
      181 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
      241 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
      301 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
      361 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
      421 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
      481 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
      541 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
      601 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
      661 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
      721 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
      781 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
      841 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
      901 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
      961 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     1021 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     1081 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     1141 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     1201 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     1261 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     1321 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     1381 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     1441 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     1501 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     1561 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     1621 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     1681 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     1741 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     1801 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     1861 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     1921 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     1981 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     2041 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     2101 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     2161 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     2221 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     2281 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     2341 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     2401 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     2461 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     2521 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     2581 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     2641 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     2701 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     2761 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     2821 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     2881 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     2941 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     3001 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     3061 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     3121 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     3181 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     3241 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     3301 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     3361 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     3421 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     3481 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     3541 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     3601 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     3661 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     3721 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     3781 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     3841 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     3901 tctagaaata attttgttta actttaagaa ggagatatac atatgggggt tggtaaaaca
     3961 tgtctcctga tatcctacac aacaaacaaa tttccatcgg agtatgtacc gactgttttt
     4021 gacaactatg cagtcacagt tatgattggt ggagaaccat atactcttgg actttttgat
     4081 actgcagggc tagaggatta tgacagatta cgaccgctga gttatccaca aacagatgta
     4141 tttctagtct gtttttcagt ggtctctcca tcttcatttg aaaacgtgaa agaaaagtgg
     4201 gtgcctgaga taactcacca ctgtccaaag actcctttct tgcttgttgg gactcaaatt
     4261 gatctcagag atgacccctc tactattgag aaacttgcca agaacaaaca gaagcctatc
     4321 actccagaga ctgctgaaaa gctggcccgt gacctgaagg ctgtcaagta tgtggagtgt
     4381 tctgcactta cacagagagg tctgaagaat gtgtttgatg aggctatcct agctgccctc
     4441 gagcctccgg aaactcaacc cggggatccc aattgggagc tcgtgtacac ggcgcgcctg
     4501 cagggaggtg gctcatctgg cggaggtcag atctcgtacg cgtcccgggg cggagtgcag
     4561 gtggaaacca tctccccagg agacgggcgc accttcccca agcgcggcca gacctgcgtg
     4621 gtgcactaca ccgggatgct tgaagatgga aagaaatttg attcctcccg ggacagaaac
     4681 aagcccttta agtttatgct aggcaagcag gaggtgatcc gaggctggga agaaggggtt
     4741 gcccagatga gtgtgggtca gagagccaaa ctgactatat ctccagatta tgcctatggt
     4801 gccactgggc acccaggcat catcccacca catgccactc tcgtcttcga tgtggagctt
     4861 ctaaaactgg aaaagcttga gaacctgtac ttccagggcg aagaaggtaa actggtaatc
     4921 tggattaacg gcgataaagg ctataacggt ctcgctgaag tcggtaagaa attcgagaaa
     4981 gataccggaa ttaaagtcac cgttgagcat ccggataaac tggaagagaa attcccacag
     5041 gttgcggcaa ctggcgatgg ccctgacatt atcttctggg cacacgaccg ctttggtggc
     5101 tacgctcaat ctggcctgtt ggctgaaatc accccggaca aagcgttcca ggacaagctg
     5161 tatccgttta cctgggatgc cgtacgttac aacggcaagc tgattgctta cccgatcgct
     5221 gttgaagcgt tatcgctgat ttataacaaa gacctgctgc cgaacccgcc aaaaacctgg
     5281 gaagagatcc cggcgctgga taaagaactg aaagcgaaag gtaagagcgc gctgatgttc
     5341 aacctgcaag aaccgtactt cacctggccg ctgattgctg ctgacggggg ttatgcgttc
     5401 aagtatgaaa acggcaagta cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa
     5461 gcgggtctga ccttcctggt tgacctgatt aaaaacaaac acatgaatgc agacaccgat
     5521 tactccatcg cagaagctgc ctttaataaa ggcgaaacag cgatgaccat caacggcccg
     5581 tgggcatggt ccaacatcga caccagcaaa gtgaattatg gtgtaacggt actgccgacc
     5641 ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc
     5701 agtccgaaca aagagctggc aaaagagttc ctcgaaaact atctgctgac tgatgaaggt
     5761 ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa
     5821 gagttggcga aagatccacg tattgccgcc actatggaaa acgcccagaa aggtgaaatc
     5881 atgccgaaca tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac
     5941 gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac taattccagc
     6001 tcgtagctcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     6061 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     6121 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     6181 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     6241 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     6301 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     6361 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     6421 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     6481 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     6541 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     6601 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     6661 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     6721 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     6781 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     6841 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     6901 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     6961 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     7021 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     7081 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     7141 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     7201 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     7261 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     7321 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     7381 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgct
//
