LOCUS       sequence-212055-        5616 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 5616)
  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 5616)
  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..5616
                     /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"
     promoter        complement(1358..1376)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1359..1376)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1434..1452)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1448..1495
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1731..1752)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    1871..1892
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(1905..2987)
                     /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     2949..2968
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2988..3065)
                     /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     3211..3230
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3386..3405)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3505..3527
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3565..3583)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3651..3755
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3756..3824,3825..4616)
                     /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      4787..5375
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5276..5295
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5529..5546
                     /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 gatccatgga tatcgaattc gtcgacctcg agggatccgg
     1321 gccctctaga tgcggccgca tgcataagct tgagtattct atagtgtcac ctaaatccca
     1381 gcttgatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     1441 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     1501 aggaggaact atatccggat aacctggcgt aatagcgaag aggcccgcac cgatcgccct
     1561 tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt aaacgttaat attttgttaa
     1621 aattcgcgtt aaatatttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     1681 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     1741 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     1801 agggcgatgg cccactacag cttgcatgcc tgcaggtcgg aagcataaag tgtaaagcct
     1861 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     1921 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     1981 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     2041 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     2101 agcaggcgaa aatcctgttt gatggtggtt gacggcggga tataacatga gctgtcttcg
     2161 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     2221 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     2281 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     2341 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     2401 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     2461 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     2521 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     2581 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     2641 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     2701 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     2761 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     2821 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     2881 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     2941 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     3001 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca ccattcgatg
     3061 gtgtcgggta ccgagctcga atttacgtga accatcaccc aaatcaagtt ttttgcggtc
     3121 gaggtgccgt aaagctctaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     3181 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     3241 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     3301 gccgctacag ggcgcgtcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3361 cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     3421 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3481 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3541 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     3601 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3661 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3721 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3781 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3841 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3901 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3961 cttttaaagt tctgctatgt catacactat tatcccgtat tgacgccggg caagagcaac
     4021 tcggtcgccg ggcgcggtat tctcagaatg acttggttga gtactcacca gtcacagaaa
     4081 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     4141 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     4201 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     4261 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     4321 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     4381 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     4441 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     4501 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     4561 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     4621 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4681 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4741 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4801 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4861 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4921 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4981 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5041 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5101 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     5161 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5221 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5281 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5341 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5401 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     5461 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     5521 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     5581 tccccgcgcg ttggccgatt cattaatgca gctgat
//
