LOCUS       sequence-422232-        6043 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express split-Rac1 fragment fused with FRB and MBP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6043)
  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 6043)
  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-Rac1/N12
FEATURES             Location/Qualifiers
     source          1..6043
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..86
                     /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             87..1169
                     /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(106..125)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1412..1417,1418..1433,1434..1440)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1448..1464
                     /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     1453..1475
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1534..2634
                     /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     2608..2631
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2683..2694
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2722..2991
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     CDS             3106..3123
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3135..3221
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3313..3340
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3368..3458
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3459..3527,3528..4319)
                     /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(3677..3696)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4407..4995
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4896..4915
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5149..5166
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5349..5371
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5365..5556)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gaaattccga caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga tagcgcccgg
       61 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
      121 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
      181 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
      241 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
      301 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
      361 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
      421 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
      481 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
      541 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
      601 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
      661 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
      721 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
      781 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
      841 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatt tcggtagtgg
      901 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg
      961 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     1021 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     1081 ccaatacaca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     1141 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact
     1201 cattaggcac aattctcatg tttgacagct tatcatcgac tgcacggtgc accaatgctt
     1261 ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat
     1321 aattcgtgtc gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata
     1381 acggttctgg caaatattct gaaatgagct gttgacaatt aatcatcggc tcgtataatg
     1441 tgtggaattg tgagcggata acaatttcac acaggaaaca gccagtccgt ttaggtgttt
     1501 tcacgagcaa ttgaccaaca aggaccatag attatgaaaa tcgaagaagg taaactggta
     1561 atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag
     1621 aaagataccg gaattaaagt caccgttgag catccggata aactggaaga gaaattccca
     1681 caggttgcgg caactggcga tggccctgac attatcttct gggcacacga ccgctttggt
     1741 ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt ccaggacaag
     1801 ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc
     1861 gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc gccaaaaacc
     1921 tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg
     1981 ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg
     2041 ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg
     2101 aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc
     2161 gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc
     2221 ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg
     2281 accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc
     2341 gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa
     2401 ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag
     2461 gaagagttgg tgaaagatcc gcgtattgcc gccactatgg aaaacgccca gaaaggtgaa
     2521 atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc
     2581 aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactaattcg
     2641 agctcgaaca acaacaacaa taacaataac aacaacctcg ggatcgaggg aaggatttca
     2701 catatgtcca tgggcggccg cgagatgtgg catgaaggcc tggaagaggc atctcgtttg
     2761 tactttgggg aaaggaacgt gaaaggcatg tttgaggtgc tggagccctt gcatgctatg
     2821 atggaacggg gcccccagac tctgaaggaa acatccttta atcaggccta tggtcgagat
     2881 ttaatggagg cccaagagtg gtgcaggaag tacatgaaat cagggaatgt caaggacctc
     2941 acccaagcct gggacctcta ttatcatgtg ttccgacgaa tctcaaagca gcagatctcg
     3001 tacgcgtccc ggggcggtgg ctcatctggc ggaggtgagc tccaggccat caagtgtgtg
     3061 gtggtgggag acggagctta aggatccgaa ttccctgcag gtaatcatca tcatcaccac
     3121 cactaaataa gcttcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     3181 tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga
     3241 acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc
     3301 atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt
     3361 cggtccgttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3421 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3481 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3541 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3601 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttc ccaatgatga
     3661 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc
     3721 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     3781 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     3841 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     3901 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     3961 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     4021 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4081 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4141 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4201 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4261 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4321 tgtcagacca agtttactca tatatacttt agattgattt ccttaggact gagcgtcaac
     4381 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4441 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4501 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     4561 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4621 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4681 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4741 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4801 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4861 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4921 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4981 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     5041 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     5101 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     5161 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5221 tcacaccgca tataaggtgc actgtgactg ggtcatggct gcgccccgac acccgccaac
     5281 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     5341 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     5401 gcagctgcgg taaagctcat cagcgtggtc gtgcagcgat tcacagatgt ctgcctgttc
     5461 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     5521 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     5581 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     5641 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     5701 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     5761 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     5821 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     5881 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     5941 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     6001 gagcacgatc atgcgcaccc gtggccagga cccaacgctg ccc
//
