LOCUS       sequence-194418-        7594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  recombinant expression of target protein as an MBP-His tag fusionat 
            the N-terminus of target protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7594)
  AUTHORS   Chung BC, Zhao J, Gillespie RA, Kwon DY, Guan Z, Hong J, Zhou P, Lee
            SY
  TITLE     Crystal structure of MraY, an essential membrane enzyme for 
            bacterial cell wall synthesis.
  JOURNAL   Science. 2013 Aug 30;341(6149):1012-1016. doi: 
            10.1126/science.1236501.
  PUBMED    23990562
REFERENCE   2  (bases 1 to 7594)
  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"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2013-08-30- 30"; volume: "341"; 
            issue: "6149"; pages: "1012-1016"
COMMENT     Sequence Label: pET26_MraYaa
FEATURES             Location/Qualifiers
     source          1..7594
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     sig_peptide     89..154
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     CDS             194..223
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             227..1327
                     /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
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1301..1324
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1337..1360
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2459..2476
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2529..2547)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2543..2590
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2627..3082
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2714..2733)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2924..2945
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3174..3989)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3174..3989)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3897..3916
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3981..4000
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4111..4699
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4600..4619
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4853..4870
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4885..5024
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4950..4969)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5110..5132
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5126..5317)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6126..7208)
                     /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     7170..7189
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7209..7286)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7492..7511
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gaaatacctg ctgccgaccg ctgctgctgg
      121 tctgctgctc ctcgctgccc agccggcgat ggcaatggat atcggaatta attcggatcc
      181 gaattcgagc tcacatcacc atcaccatca ccatcaccat caccccatga aaatcgaaga
      241 aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg
      301 taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga
      361 agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca
      421 cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc
      481 gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat
      541 tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa
      601 cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa
      661 gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga
      721 cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga
      781 taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat
      841 gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat
      901 gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt
      961 aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc
     1021 aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg aaaactatct
     1081 gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct
     1141 gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacta tggaaaacgc
     1201 ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg
     1261 tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc
     1321 gcagactccg ggtagcctgg aagttctgtt ccaggggccc gcggtaccta ggatgctgta
     1381 tcaactggct ctgctgctga aagattattg gttcgccttt aacgtcctga aatatatcac
     1441 cttccgctcg tttactgccg ttctgatcgc cttcttcctg acactggttc tgagtccgtc
     1501 ctttattaac cgtctgcgca aaatccaacg cctgtttggt ggctatgttc gtgagtatac
     1561 cccggaaagc catgaagtga aaaaatatac gccgaccatg ggtggtattg tgatcctgat
     1621 tgtggttacc ctgagcacac tgctgctgat gcgctgggat atcaaatata cctgggtggt
     1681 tctgctgtct tttctgagct ttggcaccat cggtttttgg gacgactatg tgaaactgaa
     1741 aaacaaaaaa ggcatctcca tcaaaaccaa atttctgctg caggtgctgt cagcatctct
     1801 gattagcgtg ctgatctatt attgggccga catcgacacg attctgtatt tcccgttctt
     1861 caaagagctg tatgtggatc tgggagtcct gtatctgccg ttcgctgtgt ttgtgattgt
     1921 tggtagcgcc aatgccgtca atctgaccga cgggctggat ggcctggcaa ttggaccggc
     1981 aatgactact gctactgccc tgggtgttgt tgcttatgcc gtgggacatt cgaaaatcgc
     2041 ccagtatctg aatattccgt atgttccata tgctggtgaa ctgaccgtgt tctgctttgc
     2101 tctggtgggt gctggtctgg ggtttctgtg gttcaactcc ttcccggctc aaatgtttat
     2161 gggggatgtg ggtagcctga gtattggagc gtcactggca actgtggcac tgctgaccaa
     2221 aagcgagttc atcttcgccg ttgccgctgg agtgttcgtt tttgaaacca tctccgtgat
     2281 tctgcaaatc atctatttcc gctggacagg tggaaaacgt ctgttcaaac gtgccccgtt
     2341 tcatcatcac ctggaactga atggtctgcc tgaaccgaaa attgttgtcc gtatgtggat
     2401 tattagcatc ctgctggcca ttatcgccat ttcaatgctg aaactgcgtt aactcgagca
     2461 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     2521 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     2581 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     2641 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     2701 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     2761 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     2821 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     2881 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     2941 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3001 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     3061 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     3121 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     3181 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     3241 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     3301 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     3361 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     3421 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     3481 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     3541 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     3601 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     3661 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     3721 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     3781 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     3841 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     3901 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     3961 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     4021 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4081 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4141 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4201 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     4261 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4321 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4381 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4441 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4501 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4561 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4621 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4681 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4741 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4801 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4861 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4921 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4981 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     5041 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5101 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5161 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     5221 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     5281 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     5341 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     5401 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     5461 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     5521 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     5581 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     5641 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     5701 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     5761 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     5821 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     5881 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     5941 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     6001 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     6061 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     6121 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6181 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     6241 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     6301 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     6361 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     6421 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     6481 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     6541 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     6601 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     6661 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     6721 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     6781 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     6841 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     6901 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     6961 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     7021 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     7081 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     7141 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     7201 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     7261 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     7321 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     7381 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     7441 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     7501 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     7561 ggcgtagagg atcgagatct cgatcccgcg aaat
//
