LOCUS       sequence-422225-        6049 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express split-RhoA fragment fused with FRB and MBP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6049)
  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 6049)
  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-RhoA/N12
FEATURES             Location/Qualifiers
     source          1..6049
                     /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             2763..3032
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     CDS             3153..3170
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3182..3268
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3360..3387
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3415..3505
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3506..3574,3575..4366)
                     /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(3724..3743)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4454..5042
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4943..4962
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5196..5213
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5396..5418
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5412..5603)
                     /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 acatatgtcc atgggcggcc
     2761 gcgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg gaaaggaacg
     2821 tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg ggcccccaga
     2881 ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag gcccaagagt
     2941 ggtgcaggaa gtacatgaaa tcagggaatg tcaaggacct cacccaagcc tgggacctct
     3001 attatcatgt gttccgacga atctcaaagc agcagatctc gtacgcgtcc cggggcggtg
     3061 gctcatctgg cggaggtgag ctcgctgcca tccggaagaa actggtgatt gttggtgatg
     3121 gagcctaagg atccgaattc cctgcaggta atcatcatca tcaccaccac taaataagct
     3181 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     3241 ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca
     3301 acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca
     3361 gaaggccatc ctgacggatg gcctttttgc gtttctacaa actctttcgg tccgttgttt
     3421 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     3481 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     3541 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     3601 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     3661 taagatcctt gagagttttc gccccgaaga acgtttccca atgatgagca cttttaaagt
     3721 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     3781 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     3841 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     3901 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     3961 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     4021 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     4081 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     4141 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     4201 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     4261 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     4321 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     4381 ttactcatat atactttaga ttgatttcct taggactgag cgtcaacccc gtagaaaaga
     4441 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4501 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4561 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     4621 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4681 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4741 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4801 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4861 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4921 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4981 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5041 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5101 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     5161 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     5221 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
     5281 aaggtgcact gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     5341 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5401 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     5461 agctcatcag cgtggtcgtg cagcgattca cagatgtctg cctgttcatc cgcgtccagc
     5521 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     5581 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     5641 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     5701 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     5761 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     5821 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     5881 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     5941 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     6001 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacagga
//
