LOCUS       sequence-142416-        6720 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses proteins in the periplasm as fusions to a high-binding 
            mutant (A313V) maltose-binding protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6720)
  AUTHORS   Walker IH, Hsieh PC, Riggs PD
  TITLE     Mutations in maltose-binding protein that alter affinity and 
            solubility properties.
  JOURNAL   Appl Microbiol Biotechnol. 2010 Sep;88(1):187-97. doi: 
            10.1007/s00253-010-2696-y. Epub 2010 Jun 10.
  PUBMED    20535468
REFERENCE   2  (bases 1 to 6720)
  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: 
            "10.1007/s00253-010-2696-y"; journalName: "Appl Microbiol 
            Biotechnol"; date: "2010-09"; volume: "88"; issue: "1"; pages: 
            "187-97"
COMMENT     Sequence Label: pMAL-p4X
FEATURES             Location/Qualifiers
     source          1..6720
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..80
                     /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."
     CDS             81..1163
                     /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     complement(100..119)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1406..1411,1412..1427,1428..1434)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1442..1458
                     /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)."
     primer_bind     1447..1469
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             join(1528..1605,1606..2703)
                     /codon_start=1
                     /gene="malE"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This gene encodes a signal sequence that can direct 
                     MBP to the bacterial periplasm."
                     /translation="MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNG
                     LAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAE
                     ITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDK
                     ELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLV
                     DLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPS
                     KPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKD
                     PRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2677..2700
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2752..2763
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2811..2828)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2811..2827)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2820..2842)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3006..3023)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3176..3262
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3354..3381
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3400..3491
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3492..3560,3561..4352)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3710..3729)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4394..4907)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4589..4610)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4801..4820
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5018..5606
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5507..5526
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5760..5777
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5792..5934
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5860..5879)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6020..6042
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6036..6227)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
       61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
      901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
      961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1501 gcacttcacc aacaaggacc atagcatatg aaaataaaaa caggtgcacg catcctcgca
     1561 ttatccgcat taacgacgat gatgttttcc gcctcggctc tcgccaaaat cgaagaaggt
     1621 aaactggtaa tctggattaa cggcgataaa ggctataacg gtctcgctga agtcggtaag
     1681 aaattcgaga aagataccgg aattaaagtc accgttgagc atccggataa actggaagag
     1741 aaattcccac aggttgcggc aactggcgat ggccctgaca ttatcttctg ggcacacgac
     1801 cgctttggtg gctacgctca atctggcctg ttggctgaaa tcaccccgga caaagcgttc
     1861 caggacaagc tgtatccgtt tacctgggat gccgtacgtt acaacggcaa gctgattgct
     1921 tacccgatcg ctgttgaagc gttatcgctg atttataaca aagatctgct gccgaacccg
     1981 ccaaaaacct gggaagagat cccggcgctg gataaagaac tgaaagcgaa aggtaagagc
     2041 gcgctgatgt tcaacctgca agaaccgtac ttcacctggc cgctgattgc tgctgacggg
     2101 ggttatgcgt tcaagtatga aaacggcaag tacgacatta aagacgtggg cgtggataac
     2161 gctggcgcga aagcgggtct gaccttcctg gttgacctga ttaaaaacaa acacatgaat
     2221 gcagacaccg attactccat cgcagaagct gcctttaata aaggcgaaac agcgatgacc
     2281 atcaacggcc cgtgggcatg gtccaacatc gacaccagca aagtgaatta tggtgtaacg
     2341 gtactgccga ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt
     2401 attaacgccg ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg
     2461 actgatgaag gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag
     2521 tcttacgagg aagagttggt gaaagatccg cggattgccg ccactatgga aaacgcccag
     2581 aaaggtgaaa tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact
     2641 gcggtgatca acgccgccag cggtcgtcag actgtcgatg aagccctgaa agacgcgcag
     2701 actaattcga gctcgaacaa caacaacaat aacaataaca acaacctcgg gatcgaggga
     2761 aggatttcag aattcggatc ctctagagtc gacctgcagg caagcttggc actggccgtc
     2821 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     2881 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     2941 cagttgcgca gcctgaatgg cgaatggcag cttggctgtt ttggcggatg agataagatt
     3001 ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct
     3061 ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt
     3121 agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat
     3181 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     3241 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc
     3301 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc
     3361 catcctgacg gatggccttt ttgcgtttct acaaactctt ttgtttattt ttctaaatac
     3421 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3481 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     3541 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     3601 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     3661 gttttcgccc cgaagaacgt ttcccaatga tgagcacttt taaagttctg ctatgtggcg
     3721 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     3781 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     3841 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     3901 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     3961 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4021 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4081 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     4141 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     4201 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     4261 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     4321 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     4381 tttagattga tttaccccgg ttgataatca gaaaagcccc aaaaacagga agattgtata
     4441 agcaaatatt taaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     4501 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     4561 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     4621 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     4681 accatcaccc aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     4741 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     4801 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     4861 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtaaa aggatctagg
     4921 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4981 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     5041 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     5101 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     5161 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     5221 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     5281 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5341 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     5401 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     5461 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     5521 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     5581 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5641 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     5701 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     5761 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     5821 tgtgcggtat ttcacaccgc atatatggtg cactctcagt acaatctgct ctgatgccgc
     5881 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     5941 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     6001 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     6061 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgcagcga ttcacagatg
     6121 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     6181 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     6241 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     6301 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     6361 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     6421 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     6481 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     6541 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     6601 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     6661 ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgaaatt
//
