LOCUS       sequence-422233-        6214 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express dominant negative Rac1 fused with MBP .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6214)
  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 6214)
  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-Rac1-T17N
FEATURES             Location/Qualifiers
     source          1..6214
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        50..127
                     /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             128..1210
                     /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(147..166)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1453..1458,1459..1474,1475..1481)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1489..1505
                     /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     1494..1516
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1575..2675
                     /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     2649..2672
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2724..2735
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3318..3335
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3347..3433
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3525..3552
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3580..3670
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3671..3739,3740..4531)
                     /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(3889..3908)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4619..5207
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5108..5127
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5361..5378
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5561..5583
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5577..5768)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgaaattccg acaccatcga
       61 atggtgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt
      121 ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca
      181 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
      241 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
      301 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
      361 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat
      421 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg
      481 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
      541 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag
      601 tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg
      661 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct
      721 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
      781 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
      841 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
      901 cgagtccggg ctgcgcgttg gtgcggatat ttcggtagtg ggatacgacg ataccgaaga
      961 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
     1021 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
     1081 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacac aaaccgcctc
     1141 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     1201 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca caattctcat
     1261 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc
     1321 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc
     1381 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc
     1441 tgaaatgagc tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat
     1501 aacaatttca cacaggaaac agccagtccg tttaggtgtt ttcacgagca attgaccaac
     1561 aaggaccata gattatgaaa atcgaagaag gtaaactggt aatctggatt aacggcgata
     1621 aaggctataa cggtctcgct gaagtcggta agaaattcga gaaagatacc ggaattaaag
     1681 tcaccgttga gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg
     1741 atggccctga cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc
     1801 tgttggctga aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg
     1861 atgccgtacg ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc
     1921 tgatttataa caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc
     1981 tggataaaga actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt
     2041 acttcacctg gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca
     2101 agtacgacat taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc
     2161 tggttgacct gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag
     2221 ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca
     2281 tcgacaccag caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat
     2341 ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg aacaaagagc
     2401 tggcaaaaga gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata
     2461 aagacaaacc gctgggtgcc gtagcgctga agtcttacga ggaagagttg gtgaaagatc
     2521 cgcgtattgc cgccactatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc
     2581 agatgtccgc tttctggtat gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc
     2641 agactgtcga tgaagccctg aaagacgcgc agactaattc gagctcgaac aacaacaaca
     2701 ataacaataa caacaacctc gggatcgagg gaaggatttc acatatgcag gccatcaagt
     2761 gtgtggtggt gggagacgga gctgtaggta aaaactgcct actgatcagt tacacaacca
     2821 atgcatttcc tggagaatat atccctactg tctttgacaa ttattctgcc aatgttatgg
     2881 tagatggaaa accggtgaat ctgggcttat gggatacagc tggacaagaa gattatgaca
     2941 gattacgccc cctatcctat ccgcaaacag atgtgttctt aatttgcttt tcccttgtga
     3001 gtcctgcatc atttgaaaat gtccgtgcaa agtggtatcc tgaggtgcgg caccactgtc
     3061 ccaacactcc catcatccta gtgggaacta aacttgatct tagggatgat aaagacacga
     3121 ttgagaaact gaaggagaag aagctgactc ccatcaccta tccgcagggt ctagccatgg
     3181 ctaaggagat tggtgctgta aaatacctgg agtgctcggc gctcacacag cgaggcctca
     3241 agacagtgtt tgacgaagcg atccgagcag tcctctgccc gcctcccgtg taaggatccg
     3301 aattccctgc aggtaatcat catcatcacc accactaaat aagcttcaaa taaaacgaaa
     3361 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     3421 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
     3481 gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac
     3541 ggatggcctt tttgcgtttc tacaaactct ttcggtccgt tgtttatttt tctaaataca
     3601 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3661 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3721 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3781 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3841 ttttcgcccc gaagaacgtt tcccaatgat gagcactttt aaagttctgc tatgtggcgc
     3901 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3961 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     4021 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     4081 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     4141 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     4201 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4261 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4321 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4381 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4441 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4501 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4561 ttagattgat ttccttagga ctgagcgtca accccgtaga aaagatcaaa ggatcttctt
     4621 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4681 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4741 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     4801 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4861 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4921 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4981 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5041 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5101 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5161 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5221 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     5281 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     5341 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     5401 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catataaggt gcactgtgac
     5461 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5521 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5581 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     5641 tcgtgcagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     5701 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     5761 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     5821 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     5881 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     5941 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     6001 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     6061 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     6121 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     6181 ccccgccagc ctagccgggt cctcaacgac agga
//
