LOCUS       sequence-422234-        6214 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express constitutively active 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-Q61L
FEATURES             Location/Qualifiers
     source          1..6214
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    24..40
                     /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     29..51
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             110..1210
                     /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     1184..1207
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1259..1270
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1853..1870
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1882..1968
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2060..2087
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2115..2205
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2206..2274,2275..3066)
                     /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(2424..2443)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3154..3742
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3643..3662
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3896..3913
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4096..4118
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4112..4303)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        4799..4876
                     /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             4877..5959
                     /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(4896..4915)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6202..6207,6208..6214,1..9,10..16)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
ORIGIN
        1 atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcca
       61 gtccgtttag gtgttttcac gagcaattga ccaacaagga ccatagatta tgaaaatcga
      121 agaaggtaaa ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt
      181 cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact
      241 ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc
      301 acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa
      361 agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct
      421 gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc
      481 gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg
      541 taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc
      601 tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt
      661 ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca
      721 catgaatgca gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc
      781 gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg
      841 tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag
      901 cgcaggtatt aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta
      961 tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc
     1021 gctgaagtct tacgaggaag agttggtgaa agatccgcgt attgccgcca ctatggaaaa
     1081 cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt
     1141 gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga
     1201 cgcgcagact aattcgagct cgaacaacaa caacaataac aataacaaca acctcgggat
     1261 cgagggaagg atttcacata tgcaggccat caagtgtgtg gtggtgggag acggagctgt
     1321 aggtaaaact tgcctactga tcagttacac aaccaatgca tttcctggag aatatatccc
     1381 tactgtcttt gacaattatt ctgccaatgt tatggtagat ggaaaaccgg tgaatctggg
     1441 cttatgggat acagctggac tagaagatta tgacagatta cgccccctat cctatccgca
     1501 aacagatgtg ttcttaattt gcttttccct tgtgagtcct gcatcatttg aaaatgtccg
     1561 tgcaaagtgg tatcctgagg tgcggcacca ctgtcccaac actcccatca tcctagtggg
     1621 aactaaactt gatcttaggg atgataaaga cacgatcgag aaactgaagg agaagaagct
     1681 gactcccatc acctatccgc agggtctagc catggctaag gagattggtg ctgtaaaata
     1741 cctggagtgc tcggcgctca cacagcgagg cctcaagaca gtgtttgacg aagcgatccg
     1801 agcagtcctc tgcccgcctc ccgtgtaagg atccgaattc cctgcaggta atcatcatca
     1861 tcaccaccac taaataagct tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     1921 cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg
     1981 gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact
     2041 gccaggcatc aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa
     2101 actctttcgg tccgttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     2161 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     2221 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     2281 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     2341 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgtttccca
     2401 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     2461 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     2521 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     2581 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     2641 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     2701 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     2761 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     2821 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     2881 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     2941 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     3001 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     3061 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttcct taggactgag
     3121 cgtcaacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3181 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3241 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3301 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3361 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3421 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3481 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3541 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3601 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3661 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3721 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3781 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3841 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     3901 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     3961 cggtatttca caccgcatat aaggtgcact gtgactgggt catggctgcg ccccgacacc
     4021 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     4081 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     4141 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg cagcgattca cagatgtctg
     4201 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     4261 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     4321 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     4381 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     4441 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     4501 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     4561 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     4621 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     4681 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     4741 acgacaggag cacgatcatg cgcacccgtg gccaggaccc aacgctgccc gaaattccga
     4801 caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
     4861 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
     4921 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
     4981 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
     5041 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
     5101 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
     5161 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
     5221 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
     5281 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
     5341 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     5401 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     5461 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     5521 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     5581 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     5641 cgccattacc gagtccgggc tgcgcgttgg tgcggatatt tcggtagtgg gatacgacga
     5701 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     5761 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     5821 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacaca
     5881 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     5941 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac
     6001 aattctcatg tttgacagct tatcatcgac tgcacggtgc accaatgctt ctggcgtcag
     6061 gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat aattcgtgtc
     6121 gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg
     6181 caaatattct gaaatgagct gttgacaatt aatc
//
