LOCUS       sequence-220192-        5638 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 5638)
  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 5638)
  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..5638
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     29..46
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     117..136
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        117..135
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    135..159
                     /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             172..194
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             214..231
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             241..1341
                     /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     1315..1338
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1384..1401
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1408..1428
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(1504..1522)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1505..1522)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1580..1598)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1594..1641
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1877..1898)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2048..3130)
                     /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     3092..3111
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3131..3208)
                     /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     3351..3370
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3524..3543)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3643..3665
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3703..3721)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3789..3893
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3894..3962,3963..4754)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4112..4131)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4925..5513
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5414..5433
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
       61 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctgattaat
      121 acgactcact atagggaatt gtgagcggat aacaattcct ctagaaataa ttttgtttaa
      181 ctttaagaag gagatatatc catgaacacc attcatcacc atcaccatca caacactagt
      241 atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt
      301 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat
      361 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt
      421 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc
      481 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac
      541 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa
      601 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg
      661 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg
      721 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa
      781 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt
      841 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa
      901 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa
      961 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt
     1021 ggcgtgctga gcgcaggtat tgacgccgcc agtccgaaca aagagctggc aaaagagttc
     1081 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg
     1141 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc
     1201 acaatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc
     1261 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa
     1321 gccctgaaag acgcgcagac taattcgagc tcgactagtg gatctggtgg tggtggcggc
     1381 cgcctggttc cgaggggatc catgtctgaa aacctgtact tccagggttc catggatatc
     1441 gaattcgtcg acctcgaggg atccgggccc tctagatgcg gccgcatgca taagcttgag
     1501 tattctatag tgtcacctaa atcccagctt gatccggctg ctaacaaagc ccgaaaggaa
     1561 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1621 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggataacc tggcgtaata
     1681 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     1741 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     1801 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     1861 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     1921 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca cagcttgcat gcctgcaggt
     1981 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactta cattaattgc
     2041 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     2101 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     2161 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     2221 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     2281 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     2341 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     2401 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     2461 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     2521 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     2581 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     2641 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     2701 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     2761 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     2821 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     2881 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     2941 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     3001 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     3061 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     3121 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     3181 gaaaggtttt gcaccattcg atggtgtcgg gtaccgagct cgaattgtga accatcaccc
     3241 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     3301 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     3361 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     3421 acacccgccg cgcttaatgc gccgctacag ggcgcgtcct gatgcggtat tttctcctta
     3481 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg
     3541 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3601 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     3661 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     3721 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     3781 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3841 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     3901 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     3961 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4021 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4081 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     4141 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4201 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4261 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4321 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     4381 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     4441 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     4501 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4561 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4621 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4681 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4741 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     4801 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4861 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4921 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4981 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5041 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     5101 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5161 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5221 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5281 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5341 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     5401 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5461 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5521 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     5581 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgatac
//
