LOCUS       sequence-220186-        5608 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as Thrombin cleavable 
            N-termina 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 5608)
  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 5608)
  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: pMAT9
FEATURES             Location/Qualifiers
     source          1..5608
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     39..58
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        39..57
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    57..81
                     /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             94..116
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             136..153
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             163..1263
                     /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     1237..1260
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1306..1323
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     promoter        complement(1396..1414)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1397..1414)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1472..1490)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1486..1533
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1769..1790)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1940..3022)
                     /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     2984..3003
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3023..3100)
                     /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     3243..3262
                     /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
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4004..4023)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     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 tctccccgcg cgttggccga ttcattaatg cagctgatta atacgactca ctatagggaa
       61 ttgtgagcgg ataacaattc ctctagaaat aattttgttt aactttaaga aggagatata
      121 tccatgaaca ccattcatca ccatcaccat cacaacacta gtatgaaaat cgaagaaggt
      181 aaactggtaa tctggattaa cggcgataaa ggctataacg gtctcgctga agtcggtaag
      241 aaattcgaga aagataccgg aattaaagtc accgttgagc atccggataa actggaagag
      301 aaattcccac aggttgcggc aactggcgat ggccctgaca ttatcttctg ggcacacgac
      361 cgctttggtg gctacgctca atctggcctg ttggctgaaa tcaccccgga caaagcgttc
      421 caggacaagc tgtatccgtt tacctgggat gccgtacgtt acaacggcaa gctgattgct
      481 tacccgatcg ctgttgaagc gttatcgctg atttataaca aagatctgct gccgaacccg
      541 ccaaaaacct gggaagagat cccggcgctg gataaagaac tgaaagcgaa aggtaagagc
      601 gcgctgatgt tcaacctgca agaaccgtac ttcacctggc cgctgattgc tgctgacggg
      661 ggttatgcgt tcaagtatga aaacggcaag tacgacatta aagacgtggg cgtggataac
      721 gctggcgcga aagcgggtct gaccttcctg gttgacctga ttaaaaacaa acacatgaat
      781 gcagacaccg attactccat cgcagaagct gcctttaata aaggcgaaac agcgatgacc
      841 atcaacggcc cgtgggcatg gtccaacatc gacaccagca aagtgaatta tggtgtaacg
      901 gtactgccga ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt
      961 attgacgccg ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg
     1021 actgatgaag gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag
     1081 tcttacgagg aagagttggc gaaagatcca cgtattgccg ccacaatgga aaacgcccag
     1141 aaaggtgaaa tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact
     1201 gcggtgatca acgccgccag cggtcgtcag actgtcgatg aagccctgaa agacgcgcag
     1261 actaattcga gctcgactag tggatctggt ggtggtggcg gccgcctggt tccgagggga
     1321 tccatggata tcgaattcgt cgacctcgag ggatccgggc cctctagatg cggccgcatg
     1381 cataagcttg agtattctat agtgtcacct aaatcccagc ttgatccggc tgctaacaaa
     1441 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     1501 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggataa
     1561 cctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     1621 atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     1681 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     1741 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     1801 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cacagcttgc
     1861 atgcctgcag gtcggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     1921 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     1981 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     2041 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     2101 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     2161 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     2221 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     2281 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     2341 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     2401 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     2461 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     2521 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     2581 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     2641 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     2701 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     2761 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     2821 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     2881 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     2941 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     3001 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     3061 atgccatacc gcgaaaggtt ttgcaccatt cgatggtgtc gggtaccgag ctcgaattgt
     3121 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     3181 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     3241 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     3301 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc 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 ggcgcggtat
     4021 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat 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 tgttcttcta 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 aaaccgcc
//
