LOCUS       sequence-422227-        6211 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express dominant negative RhoA fused with MBP .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6211)
  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 6211)
  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-RhoA-T19N
FEATURES             Location/Qualifiers
     source          1..6211
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        37..114
                     /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             115..1197
                     /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(134..153)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1440..1445,1446..1461,1462..1468)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1476..1492
                     /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     1481..1503
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1562..2662
                     /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     2636..2659
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2711..2722
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3302..3319
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3331..3417
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3509..3536
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3564..3654
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3655..3723,3724..4515)
                     /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(3873..3892)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4603..5191
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5092..5111
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5345..5362
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5545..5567
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5561..5752)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 cacccgtggc caggacccaa cgctgcccga aattccgaca ccatcgaatg gtgcaaaacc
       61 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
      121 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
      181 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
      241 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
      301 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
      361 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
      421 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
      481 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
      541 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
      601 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
      661 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
      721 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
      781 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
      841 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
      901 cgcgttggtg cggatatttc ggtagtggga tacgacgata ccgaagacag ctcatgttat
      961 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     1021 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
     1081 ctggtgaaaa gaaaaaccac cctggcgccc aatacacaaa ccgcctctcc ccgcgcgttg
     1141 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     1201 caacgcaatt aatgtaagtt agctcactca ttaggcacaa ttctcatgtt tgacagctta
     1261 tcatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta
     1321 tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct
     1381 ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga aatgagctgt
     1441 tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac aatttcacac
     1501 aggaaacagc cagtccgttt aggtgttttc acgagcaatt gaccaacaag gaccatagat
     1561 tatgaaaatc gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg
     1621 tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca
     1681 tccggataaa ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat
     1741 tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat
     1801 caccccggac aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta
     1861 caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa
     1921 agatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact
     1981 gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc
     2041 gctgattgct gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa
     2101 agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat
     2161 taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa
     2221 aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa
     2281 agtgaattat ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt
     2341 tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt
     2401 cctcgaaaac tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct
     2461 gggtgccgta gcgctgaagt cttacgagga agagttggtg aaagatccgc gtattgccgc
     2521 cactatggaa aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt
     2581 ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga
     2641 agccctgaaa gacgcgcaga ctaattcgag ctcgaacaac aacaacaata acaataacaa
     2701 caacctcggg atcgagggaa ggatttcaca tatggctgcc atccggaaga aactggtgat
     2761 tgttggtgat ggagcctgtg gaaagaactg cttgctcata gtcttcagca aggaccagtt
     2821 cccagaggtg tatgtgccca cagtgtttga gaactatgtg gcagatatcg aggtggatgg
     2881 aaagcaggta gagttggctt tgtgggacac agctgggcag gaagattatg atcgcctgag
     2941 gcccctctcc tacccagata ccgatgttat actgatgtgt ttttccatcg acagccctga
     3001 tagtttagaa aacatcccag aaaagtggac cccagaagtc aagcatttct gtcccaacgt
     3061 gcccatcatc ctggttggga ataagaagga tcttcggaat gatgagcaca caaggcggga
     3121 gctagccaag atgaagcagg agccggtgaa acctgaagaa ggcagagata tggcaaacag
     3181 gattggcgct tttgggtaca tggagtgttc agcaaagacc aaagatggag tgagagaggt
     3241 ttttgaaatg gctacgagag ctgctctgca agcttaagga tccgaattcc ctgcaggtaa
     3301 tcatcatcat caccaccact aaataagctt caaataaaac gaaaggctca gtcgaaagac
     3361 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg
     3421 ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg
     3481 ccataaactg ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg
     3541 tttctacaaa ctctttcggt ccgttgttta tttttctaaa tacattcaaa tatgtatccg
     3601 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     3661 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     3721 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     3781 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     3841 cgtttcccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     3901 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     3961 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     4021 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     4081 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     4141 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     4201 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     4261 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     4321 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     4381 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     4441 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     4501 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttcctt
     4561 aggactgagc gtcaaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4621 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4681 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4741 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4801 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4861 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4921 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4981 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5041 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5101 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5161 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5221 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5281 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     5341 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     5401 gcatctgtgc ggtatttcac accgcatata aggtgcactg tgactgggtc atggctgcgc
     5461 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     5521 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     5581 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtgc agcgattcac
     5641 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     5701 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     5761 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     5821 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     5881 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     5941 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     6001 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     6061 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     6121 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     6181 gggtcctcaa cgacaggagc acgatcatgc g
//
