LOCUS       sequence-147896-        5168 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses maltose binding protein (MBP) SpyCatcher fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5168)
  AUTHORS   Min D, Arbing MA, Jefferson RE, Bowie JU
  TITLE     A simple DNA handle attachment method for single molecule mechanical
            manipulation experiments.
  JOURNAL   Protein Sci. 2016 Aug;25(8):1535-44. doi: 10.1002/pro.2952. Epub 
            2016 Jun 6.
  PUBMED    27222403
REFERENCE   2  (bases 1 to 5168)
  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: "10.1002/pro.2952"; 
            journalName: "Protein Sci"; date: "2016-08"; volume: "25"; issue: 
            "8"; pages: "1535-44"
COMMENT     Sequence Label: pMBP-SpyCatcher
FEATURES             Location/Qualifiers
     source          1..5168
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             71..93
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             101..1201
                     /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
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1058..1067
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1175..1198
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1220..1237
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1259..1279
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1289..1624
                     /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="DTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMEL
                     RDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGK
                     ATKGDAHI"
     CDS             1689..1706
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1759..1777)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1773..1820
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1857..2312
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1944..1963)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2154..2175
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2338..2442
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2443..2511,2512..3303)
                     /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(2661..2680)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3474..4062
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3963..3982
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4216..4233
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4248..4387
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4313..4332)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4473..4495
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4489..4680)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg agaccacaac ggtttccctc
       61 tagaaataat tttgtttaac tttaagaagg agatatacat atgaaaatcg aagaaggtaa
      121 actggtaatc tggattaacg gcgataaagg ctataacggt ctcgctgaag tcggtaagaa
      181 attcgagaaa gataccggaa ttaaagtcac cgttgagcat ccggataaac tggaagagaa
      241 attcccacag gttgcggcaa ctggcgatgg ccctgacatt atcttctggg cacacgaccg
      301 ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc accccggaca aagcgttcca
      361 ggacaagctg tatccgttta cctgggatgc cgtacgttac aacggcaagc tgattgctta
      421 cccgatcgct gttgaagcgt tatcgctgat ttataacaaa gatctgctgc cgaacccgcc
      481 aaaaacctgg gaagagatcc cggcgctgga taaagaactg aaagcgaaag gtaagagcgc
      541 gctgatgttc aacctgcaag aaccgtactt cacctggccg ctgattgctg ctgacggggg
      601 ttatgcgttc aagtatgaaa acggcaagta cgacattaaa gacgtgggcg tggataacgc
      661 tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt aaaaacaaac acatgaatgc
      721 agacaccgat tactccatcg cagaagctgc ctttaataaa ggcgaaacag cgatgaccat
      781 caacggcccg tgggcatggt ccaacatcga caccagcaaa gtgaattatg gtgtaacggt
      841 actgccgacc ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat
      901 taacgccgcc agtccgaaca aagagctggc aaaagagttc ctcgaaaact atctgctgac
      961 tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc
     1021 ttacgaggaa gagttggcga aagatccacg tattgccgcc accatggaaa acgcccagaa
     1081 aggtgaaatc atgccgaaca tcccgcagat gtccgctttc tggtatgccg tgcgtactgc
     1141 ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac
     1201 taatggcatg tcgtactacc atcaccatca ccatcacgat tacgacatcc caacgaccga
     1261 aaacctgtat tttcagggtg gttgtggtga taccttatca ggtttatcaa gtgagcaagg
     1321 tcagtccggt gatatgacaa ttgaagaaga tagtgctacc catattaaat tctcaaaacg
     1381 tgatgaggac ggcaaagagt tagctggtgc aactatggag ttgcgtgatt catctggtaa
     1441 aactattagt acatggattt cagatggaca agtgaaagat ttctacctgt atccaggaaa
     1501 atatacattt gtcgaaaccg cagcaccaga cggttatgag gtagcaactg ctattacctt
     1561 tacagttaat gagcaaggtc aggttactgt aaatggcaaa gcaactaaag gtgacgctca
     1621 tatttaaatg gttgatgctt gaggatccga attcgagctc cgtcgacaag cttgcggccg
     1681 cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     1741 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1801 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     1861 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1921 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1981 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     2041 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     2101 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     2161 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2221 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2281 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     2341 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2401 taaccctggt aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     2461 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     2521 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     2581 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     2641 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     2701 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     2761 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     2821 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     2881 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     2941 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca
     3001 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     3061 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     3121 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     3181 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     3241 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     3301 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     3361 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     3421 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3481 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3541 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3601 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3661 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3721 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3781 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3841 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3901 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3961 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4021 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4081 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4141 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4201 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4261 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     4321 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4381 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4441 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4501 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     4561 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagcttct ccagaagcgt
     4621 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     4681 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     4741 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     4801 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     4861 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     4921 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     4981 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     5041 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5101 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac
     5161 gctgcccg
//
