LOCUS       sequence-422231-        7418 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express split-Rac1 fragment fused with FKBP and MBP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7418)
  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 7418)
  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-Rac1/13C-FKBP-MBP
FEATURES             Location/Qualifiers
     source          1..7418
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(79..126)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     122..140
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(193..210)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(235..258)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             complement(1324..1344)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1351..1671)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     RBS             2279..2301
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2316..2340
                     /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(2340..2359)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2341..2359)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2443..2462)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2668..2745
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2746..3828
                     /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(2765..2784)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4637..4828
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4822..4844)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4930..5069
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4985..5004
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5084..5101)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5255..5843)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5335..5354)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6014..6805,6806..6874))
                     /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     6637..6656
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6875..6979)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(7005..7418,1..42))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7142..7163)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7354..7373
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccaatccgga
       61 tatagttcct cctttcagca aaaaacccct caagacccgt ttagaggccc caaggggtta
      121 tgctagttat tgctcagcgg tggcagcagc caactcagct tcctttcggg ctttgttagc
      181 agccggatct cagtggtggt ggtggtggtg ctcgagctac gagctggaat tagtctgcgc
      241 gtctttcagg gcttcatcga cagtctgacg accgctggcg gcgttgatca ccgcagtacg
      301 cacggcatac cagaaagcgg acatctgcgg gatgttcggc atgatttcac ctttctgggc
      361 gttttccata gtggcggcaa tacgtggatc tttcgccaac tcttcctcgt aagacttcag
      421 cgctacggca cccagcggtt tgtctttatt aaccgcttcc agaccttcat cagtcagcag
      481 atagttttcg aggaactctt ttgccagctc tttgttcgga ctggcggcgt taatacctgc
      541 gctcagcacg ccaacgaacg gtttggatgg ttgacccttg aaggtcggca gtaccgttac
      601 accataattc actttgctgg tgtcgatgtt ggaccatgcc cacgggccgt tgatggtcat
      661 cgctgtttcg cctttattaa aggcagcttc tgcgatggag taatcggtgt ctgcattcat
      721 gtgtttgttt ttaatcaggt caaccaggaa ggtcagaccc gctttcgcgc cagcgttatc
      781 cacgcccacg tctttaatgt cgtacttgcc gttttcatac ttgaacgcat aacccccgtc
      841 agcagcaatc agcggccagg tgaagtacgg ttcttgcagg ttgaacatca gcgcgctctt
      901 acctttcgct ttcagttctt tatccagcgc cgggatctct tcccaggttt ttggcgggtt
      961 cggcagcagg tctttgttat aaatcagcga taacgcttca acagcgatcg ggtaagcaat
     1021 cagcttgccg ttgtaacgta cggcatccca ggtaaacgga tacagcttgt cctggaacgc
     1081 tttgtccggg gtgatttcag ccaacaggcc agattgagcg tagccaccaa agcggtcgtg
     1141 tgcccagaag ataatgtcag ggccatcgcc agttgccgca acctgtggga atttctcttc
     1201 cagtttatcc ggatgctcaa cggtgacttt aattccggta tctttctcga atttcttacc
     1261 gacttcagcg agaccgttat agcctttatc gccgttaatc cagattacca gtttaccttc
     1321 ttcgccctgg aagtacaggt tctcaagctt ttccagtttt agaagctcca catcgaagac
     1381 gagagtggca tgtggtggga tgatgcctgg gtgcccagtg gcaccatagg cataatctgg
     1441 agatatagtc agtttggctc tctgacccac actcatctgg gcaacccctt cttcccagcc
     1501 tcggatcacc tcctgcttgc ctagcataaa cttaaagggc ttgtttctgt cccgggagga
     1561 atcaaatttc tttccatctt caagcatccc ggtgtagtgc accacgcagg tctggccgcg
     1621 cttggggaag gtgcgcccgt ctcctgggga gatggtttcc acctgcactc cgccccggga
     1681 cgcgtacgag atctgacctc cgccagatga gccacctccc tgcaggcgcg ccgtgtacac
     1741 gagctcccaa ttgggatccc ccacgggagg cgggcagagg actgctcgga tcgcttcgtc
     1801 aaacactgtc ttgaggcctc gctgtgtgag cgccgagcac tccaggtatt ttacagcacc
     1861 aatctcctta gccatggcta gaccctgcgg ataggtgatg ggagtcagct tcttctcctt
     1921 cagtttctcg atcgtgtctt tatcatccct aagatcaagt ttagttccca ctaggatgat
     1981 gggagtgttg ggacagtggt gccgcacctc aggataccac tttgcacgga cattttcaaa
     2041 tgatgcagga ctcacaaggg aaaagcaaat taagaacaca tctgtttgcg gataggatag
     2101 ggggcgtaat ctgtcataat cttctagtcc agctgtatcc cataagccca gattcaccgg
     2161 ttttccatct accataacat tggcagaata attgtcaaag acagtaggga tatattctcc
     2221 aggaaatgca ttggttgtgt aactgatcag taggcaagtt ttacctacca tatgtatatc
     2281 tccttcttaa agttaaacaa aattatttct agaggggaat tgttatccgc tcacaattcc
     2341 cctatagtga gtcgtattaa tttcgcggga tcgagatctc gatcctctac gccggacgca
     2401 tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca
     2461 ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta
     2521 tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc
     2581 ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt
     2641 cgcataaggg agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt
     2701 atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg
     2761 ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac
     2821 caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg
     2881 aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc
     2941 gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct
     3001 cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa
     3061 gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac
     3121 tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg
     3181 ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac
     3241 ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta
     3301 gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc
     3361 actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt
     3421 tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc
     3481 aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt
     3541 gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg
     3601 ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg
     3661 caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa
     3721 agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     3781 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     3841 taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt
     3901 caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac
     3961 tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg
     4021 cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat
     4081 cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa
     4141 gcaggccatt atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt
     4201 gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg
     4261 cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat
     4321 ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat
     4381 tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt
     4441 attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac
     4501 cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg
     4561 tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc
     4621 ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat
     4681 cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc
     4741 agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg
     4801 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     4861 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     4921 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac
     4981 tatgcggcat cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca
     5041 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     5101 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5161 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5221 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5281 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5341 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5401 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5461 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5521 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5581 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5641 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     5701 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5761 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5821 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5881 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5941 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6001 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6061 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6121 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6181 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6241 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6301 atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg
     6361 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6421 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6481 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6541 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6601 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6661 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6721 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6781 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6841 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6901 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6961 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa
     7021 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     7081 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     7141 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     7201 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     7261 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     7321 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     7381 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaa
//
