LOCUS       sequence-443903-        7741 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses tandem MBPx linked to-SpyTag003 in bacterial cells. MBPx 
            is a variant of MBP (A312V I317V Delta-172-173 Delta-175-176) with 
            enhanced affinity for maltose..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7741)
  AUTHORS   Driscoll CL, Keeble AH, Howarth MR
  TITLE     SpyMask enables combinatorial assembly of bispecific binders.
  JOURNAL   Nat Commun. 2024 Mar 16;15(1):2403. doi: 10.1038/s41467-024-46599-9.
  PUBMED    38493197
REFERENCE   2  (bases 1 to 7741)
  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: "Nat 
            Commun."; date: "2024-03-16"; volume: "15(1):2403. doi"; pages: " 
            10.1038/s41467-024-46599-9"
COMMENT     Sequence Label: pET21b-MBPx-MBPx-SpyTag003
FEATURES             Location/Qualifiers
     source          1..7741
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(185..232)
                     /codon_start=1
                     /product="improved SpyTag variant that reacts with 
                     SpyCatcher003 at a rate approaching the diffusion limit 
                     (Keeble et al., 2019)"
                     /label=SpyTag003
                     /translation="RGVPHIVMVDAYKRYK"
     CDS             complement(1385..2473)
                     /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             complement(2486..2503)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(2513..2530)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2550..2572
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2587..2611
                     /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     complement(2611..2630)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2612..2630)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2714..2733)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2939..3016
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(3036..3055)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4907..5098
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5092..5114)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5200..5339
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5255..5274
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5354..5371)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5525..6113)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5605..5624)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6284..7075,7076..7144))
                     /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     6907..6926
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7145..7249)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7275..7730)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7412..7433)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7624..7643
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttct
      181 attacttgta acgcttgtag gcgtccacca tcacgatatg aggcacgcca cgaccactac
      241 cgcccgaacc cgagctcgaa ttagtctgcg cgtctttcag ggcttcatcg acagtctgac
      301 gaccgctggc ggcgttgatc accgcagtac gcacggcata ccagaaagcg gacatctgcg
      361 ggatgttcgg catgatttca cctttctggg cgttttccat agtggcggcc acacgtggat
      421 ctttcaccaa ctcttcctcg taagacttca gcgctacggc acccagcggt ttgtctttat
      481 taaccgcttc cagaccttca tcagtcagca gatagttttc gaggaactct tttgccagct
      541 ctttgttcgg actggcggcg ttaatacctg cgctcagcac gccaacgaac ggtttggatg
      601 gttgaccctt gaaggtcggc agtaccgtta caccataatt cactttgctg gtgtcgatgt
      661 tggaccatgc ccacgggccg ttgatggtca tcgctgtttc gcctttatta aaggcagctt
      721 ctgcgatgga gtaatcggtg tctgcattca tgtgtttgtt tttaatcagg tcaaccagga
      781 aggtcagacc cgctttcgcg ccagcgttat ccacgcccac gtctttaatg tcgccatact
      841 tgaacgcata acccccgtca gcagcaatca gcggccaggt gaagtacggt tcttgcaggt
      901 tgaacatcag cgcgctctta cctttcgctt tcagttcttt atccagcgcc gggatctctt
      961 cccaggtttt tggcgggttc ggcagcagat ctttgttata aatcagcgat aacgcttcaa
     1021 cagcgatcgg gtaagcaatc agcttgccgt tgtaacgtac ggcatcccag gtaaacggat
     1081 acagcttgtc ctggaacgct ttgtccgggg tgatttcagc caacaggcca gattgagcgt
     1141 agccaccaaa gcggtcgtgt gcccagaaga taatgtcagg gccatcgcca gttgccgcaa
     1201 cctgtgggaa tttctcttcc agtttatccg gatgctcaac ggtgacttta attccggtat
     1261 ctttctcgaa tttcttaccg acttcagcga gaccgttata gcctttatcg ccgttaatcc
     1321 agattaccag tttaccctcc tctatctttc cggatccacc tccggatccg ccggaactag
     1381 aattcgtctg cgcgtctttc agggcttcat cgacagtctg acgaccgctg gcggcgttga
     1441 tcaccgcagt acgcacggca taccagaaag cggacatctg cgggatgttc ggcatgattt
     1501 cacctttctg ggcgttttcc atagtggcgg ccacacgtgg atctttcacc aactcttcct
     1561 cgtaagactt cagcgctacg gcacccagcg gtttgtcttt attaaccgct tccagacctt
     1621 catcagtcag cagatagttt tcgaggaact cttttgccag ctctttgttc ggactggcgg
     1681 cgttaatacc tgcgctcagc acgccaacga acggtttgga tggttgaccc ttgaaggtcg
     1741 gcagtaccgt tacaccataa ttcactttgc tggtgtcgat gttggaccat gcccacgggc
     1801 cgttgatggt catcgctgtt tcgcctttat taaaggcagc ttctgcgatg gagtaatcgg
     1861 tgtctgcatt catgtgtttg tttttaatca ggtcaaccag gaaggtcaga cccgctttcg
     1921 cgccagcgtt atccacgccc acgtctttaa tgtcgccata cttgaacgca taacccccgt
     1981 cagcagcaat cagcggccag gtgaagtacg gttcttgcag gttgaacatc agcgcgctct
     2041 tacctttcgc tttcagttct ttatccagcg ccgggatctc ttcccaggtt tttggcgggt
     2101 tcggcagcag atctttgtta taaatcagcg ataacgcttc aacagcgatc gggtaagcaa
     2161 tcagcttgcc gttgtaacgt acggcatccc aggtaaacgg atacagcttg tcctggaacg
     2221 ctttgtccgg ggtgatttca gccaacaggc cagattgagc gtagccacca aagcggtcgt
     2281 gtgcccagaa gataatgtca gggccatcgc cagttgccgc aacctgtggg aatttctctt
     2341 ccagtttatc cggatgctca acggtgactt taattccggt atctttctcg aatttcttac
     2401 cgacttcagc gagaccgtta tagcctttat cgccgttaat ccagattacc agtttacctt
     2461 cttcgatttt catgctagcc atatggctgc cgcgcggcac caggccgctg ctgtgatgat
     2521 gatgatgatg gctgctgccc atatgtatat ctccttctta aagttaaaca aaattatttc
     2581 tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg
     2641 atcgagatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt
     2701 gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc
     2761 gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg
     2821 ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac
     2881 ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga
     2941 caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
     3001 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
     3061 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
     3121 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
     3181 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
     3241 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
     3301 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
     3361 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
     3421 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
     3481 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     3541 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     3601 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     3661 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     3721 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     3781 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
     3841 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg atttcgcctg
     3901 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc
     3961 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa
     4021 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     4081 ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc
     4141 gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg
     4201 gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca
     4261 ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat
     4321 gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac
     4381 tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggcccc
     4441 acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct
     4501 tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa
     4561 aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct
     4621 ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg
     4681 ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgtgt
     4741 gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag
     4801 taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg
     4861 tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga
     4921 aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa
     4981 actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc
     5041 tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     5101 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     5161 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     5221 tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga
     5281 gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     5341 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5401 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5461 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5521 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5581 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5641 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5701 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5761 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5821 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5881 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5941 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     6001 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6061 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6121 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6181 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6241 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6301 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6361 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6421 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6481 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6541 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg
     6601 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6661 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6721 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6781 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6841 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6901 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6961 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     7021 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     7081 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7141 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     7201 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7261 gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     7321 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     7381 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     7441 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     7501 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     7561 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     7621 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     7681 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc
     7741 a
//
