LOCUS       sequence-422224-        7409 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express split-RhoA fragment fused with FKBP and MBP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7409)
  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 7409)
  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: pET21b-RhoA/13C-FKBP-MBP
FEATURES             Location/Qualifiers
     source          1..7409
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(19..66)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     62..80
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(133..150)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(175..198)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             complement(1264..1284)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1291..1611)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     RBS             2210..2232
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2247..2271
                     /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     complement(2271..2290)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2272..2290)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2374..2393)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2599..2676
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2677..3759
                     /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(2696..2715)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4568..4759
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4753..4775)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4861..5000
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4916..4935
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5015..5032)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5186..5774)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5266..5285)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5945..6736,6737..6805))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6568..6587
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6806..6910)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6936..7391)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7073..7094)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7285..7304
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tatagttcct cctttcagca aaaaacccct caagacccgt ttagaggccc caaggggtta
       61 tgctagttat tgctcagcgg tggcagcagc caactcagct tcctttcggg ctttgttagc
      121 agccggatct cagtggtggt ggtggtggtg ctcgagctac gagctggaat tagtctgcgc
      181 gtctttcagg gcttcatcga cagtctgacg accgctggcg gcgttgatca ccgcagtacg
      241 cacggcatac cagaaagcgg acatctgcgg gatgttcggc atgatttcac ctttctgggc
      301 gttttccata gtggcggcaa tacgtggatc tttcgccaac tcttcctcgt aagacttcag
      361 cgctacggca cccagcggtt tgtctttatt aaccgcttcc agaccttcat cagtcagcag
      421 atagttttcg aggaactctt ttgccagctc tttgttcgga ctggcggcgt taatacctgc
      481 gctcagcacg ccaacgaacg gtttggatgg ttgacccttg aaggtcggca gtaccgttac
      541 accataattc actttgctgg tgtcgatgtt ggaccatgcc cacgggccgt tgatggtcat
      601 cgctgtttcg cctttattaa aggcagcttc tgcgatggag taatcggtgt ctgcattcat
      661 gtgtttgttt ttaatcaggt caaccaggaa ggtcagaccc gctttcgcgc cagcgttatc
      721 cacgcccacg tctttaatgt cgtacttgcc gttttcatac ttgaacgcat aacccccgtc
      781 agcagcaatc agcggccagg tgaagtacgg ttcttgcagg ttgaacatca gcgcgctctt
      841 acctttcgct ttcagttctt tatccagcgc cgggatctct tcccaggttt ttggcgggtt
      901 cggcagcagg tctttgttat aaatcagcga taacgcttca acagcgatcg ggtaagcaat
      961 cagcttgccg ttgtaacgta cggcatccca ggtaaacgga tacagcttgt cctggaacgc
     1021 tttgtccggg gtgatttcag ccaacaggcc agattgagcg tagccaccaa agcggtcgtg
     1081 tgcccagaag ataatgtcag ggccatcgcc agttgccgca acctgtggga atttctcttc
     1141 cagtttatcc ggatgctcaa cggtgacttt aattccggta tctttctcga atttcttacc
     1201 gacttcagcg agaccgttat agcctttatc gccgttaatc cagattacca gtttaccttc
     1261 ttcgccctgg aagtacaggt tctcaagctt ttccagtttt agaagctcca catcgaagac
     1321 gagagtggca tgtggtggga tgatgcctgg gtgcccagtg gcaccatagg cataatctgg
     1381 agatatagtc agtttggctc tctgacccac actcatctgg gcaacccctt cttcccagcc
     1441 tcggatcacc tcctgcttgc ctagcataaa cttaaagggc ttgtttctgt cccgggagga
     1501 atcaaatttc tttccatctt caagcatccc ggtgtagtgc accacgcagg tctggccgcg
     1561 cttggggaag gtgcgcccgt ctcctgggga gatggtttcc acctgcactc cgccccggga
     1621 cgcgtacgag atctgacctc cgccagatga gccacctccc tgcaggcgcg ccgtgtacac
     1681 gagctcccaa ttgggatccc cagcttgcag agcagctctc gtagccattt caaaaacctc
     1741 tctcactcca tctttggtct ttgctgaaca ctccatgtac ccaaaagcgc caatcctgtt
     1801 tgccatatct ctgccttctt caggtttcac cggctcctgc ttcatcttgg ctagctcccg
     1861 ccttgtgtgc tcatcattcc gaagatcctt cttattccca accaggatga tgggcacgtt
     1921 gggacagaaa tgcttgactt ctggggtcca cttttctggg atgttttcta aactatcagg
     1981 gctgtcgatg gaaaaacaca tcagtataac atcggtatct gggtaggaga ggggcctcag
     2041 gcgatcataa tcttccagcc cagctgtgtc ccacaaagcc aactctacct gctttccatc
     2101 cacctcgata tctgccacat agttctcaaa cactgtgggc acatacacct ctgggaactg
     2161 atccttgctg aagactatga gcaagcatgt ctttccacac atatgtatat ctccttctta
     2221 aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg
     2281 agtcgtatta atttcgcggg atcgagatct cgatcctcta cgccggacgc atcgtggccg
     2341 gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg
     2401 aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag
     2461 gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg
     2521 cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg
     2581 gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga
     2641 tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat
     2701 gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc
     2761 cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt
     2821 cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc
     2881 tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat
     2941 caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa
     3001 gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg
     3061 gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt
     3121 gatgtctctg accagacacc catcaacagt attattttct cccatgaaga cggtacgcga
     3181 ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca
     3241 ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat
     3301 caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa
     3361 accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag
     3421 atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc
     3481 tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc
     3541 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct
     3601 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc
     3661 accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     3721 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag
     3781 ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt
     3841 cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt
     3901 tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg
     3961 ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc
     4021 cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat
     4081 tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg
     4141 gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac
     4201 gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg
     4261 tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg
     4321 gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa
     4381 gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg
     4441 tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag
     4501 catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga
     4561 ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca
     4621 gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg
     4681 tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga
     4741 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     4801 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     4861 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
     4921 tcagagcaga ttgtactgag agtgcaccat tgcggtgtga aataccgcac agatgcgtaa
     4981 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5041 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5101 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5161 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5221 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5281 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5341 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5401 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5461 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5521 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5581 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     5641 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5701 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5761 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5821 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5881 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5941 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6001 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6061 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6121 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6181 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6241 gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6301 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6361 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6421 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6481 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6541 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6601 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6661 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6721 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6781 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6841 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6901 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     6961 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     7021 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     7081 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     7141 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     7201 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     7261 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     7321 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     7381 acagggcgcg tcccattcgc caatccgga
//
