LOCUS       sequence-433206-        4133 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyCatcher003-TEV site-SpyTag003DA in bacterial cytoplasm,
            to allow TEV protease to remove SpyTag003DA and thereby uncage 
            SpyCatcher003 reactivity..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4133)
  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 4133)
  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: pDEST14-SpyCatcher003-TEVs-SpyTag003DA
FEATURES             Location/Qualifiers
     source          1..4133
                     /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             113..130
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             509..529
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             542..589
                     /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="RGVPHIVMVAAYKRYK"
     CDS             654..671
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(724..742)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      738..785
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      822..1277
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(909..928)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1119..1140
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1303..1407
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1408..1476,1477..2268)
                     /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(1626..1645)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2439..3027
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2928..2947
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3181..3198
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3213..3352
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3278..3297)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3438..3460
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3454..3645)
                     /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 atgtcgtact accatcacca
      121 tcaccatcac gattacgaca tcccaacgac cggcgccatg gtaaccacct tatcaggttt
      181 atcaggtgag caaggtccgt ccggtgatat gacaactgaa gaagatagtg ctacccatat
      241 taaattctca aaacgtgatg aggacggccg tgagttagct ggtgcaacta tggagttgcg
      301 tgattcatct ggtaaaacta ttagtacatg gatttcagat ggacatgtga aggatttcta
      361 cctgtatcca ggaaaatata catttgtcga aaccgcagca ccagacggtt atgaggtagc
      421 aactccaatt gaatttacag ttaatgagga cggtcaggtt actgtagatg gtgaagcaac
      481 tgaaggtgac gctggatcca gtggtagcga aaacctgtat tttcagggcg gtggtagcgg
      541 ccgtggcgtt cctcatattg ttatggtggc cgcctacaaa cgctataaat aaatggttga
      601 tgcttgagga tccgaattcg agctccgtcg acaagcttgc ggccgcactc gagcaccacc
      661 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
      721 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
      781 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
      841 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
      901 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
      961 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     1021 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     1081 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     1141 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     1201 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     1261 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1321 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctggtaaatg
     1381 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1441 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     1501 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     1561 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     1621 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     1681 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     1741 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     1801 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     1861 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     1921 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     1981 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     2041 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     2101 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     2161 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     2221 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     2281 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     2341 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2401 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2461 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2521 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2581 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2641 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2701 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     2761 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2821 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2881 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2941 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3001 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3061 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3121 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     3181 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     3241 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     3301 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     3361 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     3421 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     3481 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     3541 tgcctgttca tccgcgtcca gctcgttgag cttctccaga agcgttaatg tctggcttct
     3601 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     3661 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     3721 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     3781 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     3841 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     3901 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     3961 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     4021 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     4081 caacgacagg agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccg
//
