LOCUS       sequence-422216-        6557 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provide a bacterial expression vector that encodes C-terminus MBP 
            tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6557)
  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 6557)
  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-C-MBP
FEATURES             Location/Qualifiers
     source          1..6557
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(238..1029,1030..1098))
                     /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     861..880
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1099..1203)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1229..1684)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1366..1387)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1578..1597
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1721..1768)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1764..1782
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1835..1852)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1877..1900)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             complement(2966..2986)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(3020..3052)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     RBS             3060..3082
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3097..3121
                     /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(3121..3140)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3122..3140)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3224..3243)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3449..3526
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3527..4609
                     /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(3546..3565)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5418..5609
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5603..5625)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5711..5850
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5766..5785
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5865..5882)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6036..6557,1..67))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6116..6135)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
       61 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
      121 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
      181 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
      241 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
      301 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
      361 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
      421 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
      481 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
      541 tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
      601 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
      661 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
      721 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
      781 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
      841 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
      901 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
      961 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     1021 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     1081 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     1141 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     1201 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     1261 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     1321 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     1381 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     1441 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     1501 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     1561 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     1621 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     1681 gcgtcccatt cgccaatccg gatatagttc ctcctttcag caaaaaaccc ctcaagaccc
     1741 gtttagaggc cccaaggggt tatgctagtt attgctcagc ggtggcagca gccaactcag
     1801 cttcctttcg ggctttgtta gcagccggat ctcagtggtg gtggtggtgg tgctcgagct
     1861 acgagctgga attagtctgc gcgtctttca gggcttcatc gacagtctga cgaccgctgg
     1921 cggcgttgat caccgcagta cgcacggcat accagaaagc ggacatctgc gggatgttcg
     1981 gcatgatttc acctttctgg gcgttttcca tagtggcggc aatacgtgga tctttcgcca
     2041 actcttcctc gtaagacttc agcgctacgg cacccagcgg tttgtcttta ttaaccgctt
     2101 ccagaccttc atcagtcagc agatagtttt cgaggaactc ttttgccagc tctttgttcg
     2161 gactggcggc gttaatacct gcgctcagca cgccaacgaa cggtttggat ggttgaccct
     2221 tgaaggtcgg cagtaccgtt acaccataat tcactttgct ggtgtcgatg ttggaccatg
     2281 cccacgggcc gttgatggtc atcgctgttt cgcctttatt aaaggcagct tctgcgatgg
     2341 agtaatcggt gtctgcattc atgtgtttgt ttttaatcag gtcaaccagg aaggtcagac
     2401 ccgctttcgc gccagcgtta tccacgccca cgtctttaat gtcgtacttg ccgttttcat
     2461 acttgaacgc ataacccccg tcagcagcaa tcagcggcca ggtgaagtac ggttcttgca
     2521 ggttgaacat cagcgcgctc ttacctttcg ctttcagttc tttatccagc gccgggatct
     2581 cttcccaggt ttttggcggg ttcggcagca ggtctttgtt ataaatcagc gataacgctt
     2641 caacagcgat cgggtaagca atcagcttgc cgttgtaacg tacggcatcc caggtaaacg
     2701 gatacagctt gtcctggaac gctttgtccg gggtgatttc agccaacagg ccagattgag
     2761 cgtagccacc aaagcggtcg tgtgcccaga agataatgtc agggccatcg ccagttgccg
     2821 caacctgtgg gaatttctct tccagtttat ccggatgctc aacggtgact ttaattccgg
     2881 tatctttctc gaatttctta ccgacttcag cgagaccgtt atagccttta tcgccgttaa
     2941 tccagattac cagtttacct tcttcgccct ggaagtacag gttctcaagc ttgtcgacgg
     3001 agctcgaatt cggatcccga cccatttgct gtccaccagt catgctagcc atatgtatat
     3061 ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc
     3121 ccctatagtg agtcgtatta atttcgcggg atcgagatct cgatcctcta cgccggacgc
     3181 atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc
     3241 accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt
     3301 atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc
     3361 cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag
     3421 tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg
     3481 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
     3541 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     3601 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     3661 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     3721 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     3781 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     3841 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     3901 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     3961 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
     4021 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     4081 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     4141 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     4201 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     4261 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     4321 tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
     4381 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     4441 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
     4501 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4561 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     4621 ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct
     4681 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
     4741 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg
     4801 gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa
     4861 tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga
     4921 agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt
     4981 tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac
     5041 gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa
     5101 tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca
     5161 ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg
     5221 tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata
     5281 ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc
     5341 gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc
     5401 cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta
     5461 tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg
     5521 cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc
     5581 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     5641 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     5701 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
     5761 ctatgcggca tcagagcaga ttgtactgag agtgcaccat tgcggtgtga aataccgcac
     5821 agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg
     5881 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5941 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     6001 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     6061 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     6121 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     6181 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     6241 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6301 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6361 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6421 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     6481 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6541 tgatccggca aacaaac
//
