LOCUS       sequence-422229-        6211 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express constitutively active 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-Q63L
FEATURES             Location/Qualifiers
     source          1..6211
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        19..96
                     /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             97..1179
                     /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(116..135)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1422..1427,1428..1443,1444..1450)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1458..1474
                     /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     1463..1485
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1544..2644
                     /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     2618..2641
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2693..2704
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3284..3301
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3313..3399
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3491..3518
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3546..3636
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3637..3705,3706..4497)
                     /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(3855..3874)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4585..5173
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5074..5093
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5327..5344
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5527..5549
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5543..5734)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 aacgctgccc gaaattccga caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga
       61 tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat
      121 gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc
      181 cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt
      241 cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc
      301 tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat
      361 caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa
      421 gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg
      481 gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt
      541 gatgtctctg accagacacc catcaacagt attattttct cccatgaaga cggtacgcga
      601 ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca
      661 ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat
      721 caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa
      781 accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag
      841 atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatt
      901 tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc
      961 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct
     1021 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc
     1081 accctggcgc ccaatacaca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     1141 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag
     1201 ttagctcact cattaggcac aattctcatg tttgacagct tatcatcgac tgcacggtgc
     1261 accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa
     1321 atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg ttttttgcgc
     1381 cgacatcata acggttctgg caaatattct gaaatgagct gttgacaatt aatcatcggc
     1441 tcgtataatg tgtggaattg tgagcggata acaatttcac acaggaaaca gccagtccgt
     1501 ttaggtgttt tcacgagcaa ttgaccaaca aggaccatag attatgaaaa tcgaagaagg
     1561 taaactggta atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa
     1621 gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata aactggaaga
     1681 gaaattccca caggttgcgg caactggcga tggccctgac attatcttct gggcacacga
     1741 ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt
     1801 ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc
     1861 ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc
     1921 gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag
     1981 cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg
     2041 gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa
     2101 cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa
     2161 tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac
     2221 catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac
     2281 ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg
     2341 tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct
     2401 gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa
     2461 gtcttacgag gaagagttgg tgaaagatcc gcgtattgcc gccactatgg aaaacgccca
     2521 gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac
     2581 tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca
     2641 gactaattcg agctcgaaca acaacaacaa taacaataac aacaacctcg ggatcgaggg
     2701 aaggatttca catatggctg ccatccggaa gaaactggtg attgttggtg atggagcctg
     2761 tggaaagaca tgcttgctca tagtcttcag caaggaccag ttcccagagg tgtatgtgcc
     2821 cacagtgttt gagaactatg tggcagatat cgaggtggat ggaaagcagg tagagttggc
     2881 tttgtgggac acagctgggc tggaagatta tgatcgcctg aggcccctct cctacccaga
     2941 taccgatgtt atactgatgt gtttttccat cgacagccct gatagtttag aaaacatccc
     3001 agaaaagtgg accccagaag tcaagcattt ctgtcccaac gtgcccatca tcctggttgg
     3061 gaataagaag gatcttcgga atgatgagca cacaaggcgg gagctagcca agatgaagca
     3121 ggagccggtg aaacctgaag aaggcagaga tatggcaaac aggattggcg cttttgggta
     3181 catggagtgt tcagcaaaga ccaaagatgg agtgagagag gtttttgaaa tggctacgag
     3241 agctgctctg caagcttaag gatccgaatt ccctgcaggt aatcatcatc atcaccacca
     3301 ctaaataagc ttcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
     3361 tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac
     3421 gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat
     3481 caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca aactctttcg
     3541 gtccgttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3601 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3661 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3721 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3781 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgtttccc aatgatgagc
     3841 acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa
     3901 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3961 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     4021 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     4081 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     4141 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     4201 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     4261 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     4321 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4381 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     4441 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4501 tcagaccaag tttactcata tatactttag attgatttcc ttaggactga gcgtcaaccc
     4561 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4621 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4681 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     4741 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4801 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4861 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4921 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4981 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     5041 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     5101 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     5161 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     5221 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     5281 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     5341 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     5401 acaccgcata taaggtgcac tgtgactggg tcatggctgc gccccgacac ccgccaacac
     5461 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     5521 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     5581 agctgcggta aagctcatca gcgtggtcgt gcagcgattc acagatgtct gcctgttcat
     5641 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     5701 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     5761 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     5821 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     5881 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     5941 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     6001 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     6061 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     6121 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     6181 gcacgatcat gcgcacccgt ggccaggacc c
//
