LOCUS       sequence-434061-        8038 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of tandem high affinity maltose binding protein
            domains linked to SpyCatcher, for solid-phase polyprotein 
            synthesis..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8038)
  AUTHORS   Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, 
            Howarth M
  TITLE     Programmable polyproteams built using twin peptide superglues.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 Jan 19. pii: 201519214.
  PUBMED    26787909
REFERENCE   2  (bases 1 to 8038)
  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: "Proc Natl 
            Acad Sci U S A. 2016 Jan 19. pii: 201519214."
COMMENT     Sequence Label: pET21 MBPx-SpyCatcher
FEATURES             Location/Qualifiers
     source          1..8038
                     /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    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             101..118
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             128..145
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             158..1246
                     /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
                     ADGGYAFKYGDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMT
                     INGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYL
                     LTDEGLEAVNKDKPLGAVALKSYEEELVKDPRVAATMENAQKGEIMPNIPQMSAFWYAV
                     RTAVINAASGRQTVDEALKDAQT"
     CDS             2405..2743
                     /codon_start=1
                     /product="truncated version of the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyCatcher
                     /note="forms a covalent isopeptide bond with the SpyTag 
                     peptide (Zakeri et al., 2012)"
                     /translation="VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATME
                     LRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     KATKGDAHI"
     CDS             2771..2788
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2841..2859)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2855..2902
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2939..3394
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3026..3045)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3236..3257
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3420..3524
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3525..3593,3594..4385)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3743..3762)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4556..5144
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5045..5064
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5298..5315
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5330..5469
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5395..5414)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5555..5577
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5571..5762)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     7614..7633
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7653..7730)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7936..7955
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gggcagcagc catcatcatc atcatcacag
      121 cagcggcctg gtgccgcgcg gcagccatat ggctagcatg aaaatcgaag aaggtaaact
      181 ggtaatctgg attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt
      241 cgagaaagat accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt
      301 cccacaggtt gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt
      361 tggtggctac gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga
      421 caagctgtat ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc
      481 gatcgctgtt gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa
      541 aacctgggaa gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct
      601 gatgttcaac ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta
      661 tgcgttcaag tatggcgaca ttaaagacgt gggcgtggat aacgctggcg cgaaagcggg
      721 tctgaccttc ctggttgacc tgattaaaaa caaacacatg aatgcagaca ccgattactc
      781 catcgcagaa gctgccttta ataaaggcga aacagcgatg accatcaacg gcccgtgggc
      841 atggtccaac atcgacacca gcaaagtgaa ttatggtgta acggtactgc cgaccttcaa
      901 gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg ccgccagtcc
      961 gaacaaagag ctggcaaaag agttcctcga aaactatctg ctgactgatg aaggtctgga
     1021 agcggttaat aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg aggaagagtt
     1081 ggtgaaagat ccacgtgtgg ccgccactat ggaaaacgcc cagaaaggtg aaatcatgcc
     1141 gaacatcccg cagatgtccg ctttctggta tgccgtgcgt actgcggtga tcaacgccgc
     1201 cagcggtcgt cagactgtcg atgaagccct gaaagacgcg cagacgaatt ctagttccgg
     1261 cggatccgga ggtggatccg gaaagataga ggagggtaaa ctggtaatct ggattaacgg
     1321 cgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat
     1381 taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac
     1441 tggcgatggc cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc
     1501 tggcctgttg gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac
     1561 ctgggatgcc gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt
     1621 atcgctgatt tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc
     1681 ggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga
     1741 accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatggcga
     1801 cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga
     1861 cctgattaaa aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt
     1921 taataaaggc gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac
     1981 cagcaaagtg aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc
     2041 gttcgttggc gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcaaa
     2101 agagttcctc gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa
     2161 accgctgggt gccgtagcgc tgaagtctta cgaggaagag ttggtgaaag atccacgtgt
     2221 ggccgccact atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc
     2281 cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc gtcagactgt
     2341 cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg ggttcgggcg gtagtggtgc
     2401 catggttgat accttatcag gtttatcaag tgagcaaggt cagtccggtg atatgacaat
     2461 tgaagaagat agtgctaccc atattaaatt ctcaaaacgt gatgaggacg gcaaagagtt
     2521 agctggtgca actatggagt tgcgtgattc atctggtaaa actattagta catggatttc
     2581 agatggacaa gtgaaagatt tctacctgta tccaggaaaa tatacatttg tcgaaaccgc
     2641 agcaccagac ggttatgagg tagcaactgc tattaccttt acagttaatg agcaaggtca
     2701 ggttactgta aatggcaaag caactaaagg tgacgctcat atttaataga agcttgcggc
     2761 cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
     2821 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     2881 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     2941 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3001 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3061 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3121 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     3181 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     3241 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     3301 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     3361 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     3421 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3481 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     3541 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3601 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3661 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3721 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     3781 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3841 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3901 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3961 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     4021 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     4081 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     4141 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     4201 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     4261 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     4321 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     4381 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     4441 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4501 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4561 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4621 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4681 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     4741 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4801 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4861 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4921 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4981 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5041 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5101 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5161 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5221 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5281 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     5341 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     5401 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     5461 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5521 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5581 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     5641 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     5701 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     5761 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     5821 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     5881 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac acagggtcaa
     5941 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     6001 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     6061 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     6121 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     6181 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     6241 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     6301 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     6361 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     6421 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     6481 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     6541 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     6601 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     6661 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     6721 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaatcc
     6781 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     6841 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     6901 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     6961 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     7021 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     7081 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     7141 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     7201 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     7261 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     7321 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     7381 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     7441 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     7501 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     7561 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     7621 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     7681 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     7741 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     7801 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     7861 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     7921 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     7981 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaat
//
