LOCUS       sequence-212057-        5646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as Thrombin and TEV 
            cleavableN-terminal 6His-tag-MBP fusions and without NcoI site 
            within MBP. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5646)
  AUTHORS   Peranen J, Rikkonen M, Hyvonen M, Kaariainen L
  TITLE     T7 vectors with modified T7lac promoter for expression of proteins 
            in Escherichia coli.
  JOURNAL   Anal Biochem. 1996 May 1;236(2):371-3.
  PUBMED    8660525
REFERENCE   2  (bases 1 to 5646)
  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: "Anal 
            Biochem."; date: "1996-05-1"; volume: "236(2)"; pages: "371-3"
COMMENT     Sequence Label: pMAT11
FEATURES             Location/Qualifiers
     source          1..5646
                     /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    19..43
                     /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             56..78
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             98..115
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             125..1225
                     /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
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGIDAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1199..1222
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1268..1285
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1292..1312
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(1388..1406)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1389..1406)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1464..1482)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1478..1525
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1761..1782)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    1901..1922
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(1935..3017)
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2979..2998
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3018..3095)
                     /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."
     primer_bind     3241..3260
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3416..3435)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3535..3557
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3595..3613)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3681..3785
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3786..3854,3855..4646)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCHTLLSRIDAGQEQLGRRARYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4817..5405
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5306..5325
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5559..5576
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa caccattcat caccatcacc atcacaacac
      121 tagtatgaaa atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa
      181 cggtctcgct gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga
      241 gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg atggccctga
      301 cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga
      361 aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg
      421 ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa
      481 caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga
      541 actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg
      601 gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat
      661 taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct
      721 gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa
      781 taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag
      841 caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt
      901 cgttggcgtg ctgagcgcag gtattgacgc cgccagtccg aacaaagagc tggcaaaaga
      961 gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc
     1021 gctgggtgcc gtagcgctga agtcttacga ggaagagttg gcgaaagatc cacgtattgc
     1081 cgccacaatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc
     1141 tttctggtat gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc agactgtcga
     1201 tgaagccctg aaagacgcgc agactaattc gagctcgact agtggatctg gtggtggtgg
     1261 cggccgcctg gttccgaggg gatccatgtc tgaaaacctg tacttccagg gttccatgga
     1321 tatcgaattc gtcgacctcg agggatccgg gccctctaga tgcggccgca tgcataagct
     1381 tgagtattct atagtgtcac ctaaatccca gcttgatccg gctgctaaca aagcccgaaa
     1441 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1501 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat aacctggcgt
     1561 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     1621 tggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatatttgt taaatcagct
     1681 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     1741 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     1801 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacag cttgcatgcc
     1861 tgcaggtcgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     1921 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     1981 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     2041 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     2101 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     2161 gacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     2221 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     2281 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     2341 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     2401 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     2461 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     2521 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     2581 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     2641 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     2701 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     2761 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     2821 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     2881 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     2941 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     3001 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     3061 ataccgcgaa aggttttgca ccattcgatg gtgtcgggta ccgagctcga atttacgtga
     3121 accatcaccc aaatcaagtt ttttgcggtc gaggtgccgt aaagctctaa atcggaaccc
     3181 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     3241 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     3301 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcgc ctgatgcggt
     3361 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
     3421 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     3481 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3541 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     3601 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     3661 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3721 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3781 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     3841 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     3901 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     3961 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt catacactat
     4021 tatcccgtat tgacgccggg caagagcaac tcggtcgccg ggcgcggtat tctcagaatg
     4081 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4141 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4201 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4261 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     4321 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     4381 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     4441 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4501 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     4561 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     4621 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     4681 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     4741 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4801 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4861 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     4921 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     4981 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5041 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     5101 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5161 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5221 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     5281 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     5341 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     5401 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     5461 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     5521 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     5581 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     5641 gctgat
//
