LOCUS       sequence-433325-        4520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyCatcher003-SpyCatcher003-TEV site-SpyTag003DA in 
            bacterial cytoplasm, to allow stepwise coupling of two unique 
            SpyTag-linked binders.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4520)
  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 4520)
  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-DoubleCatcher
FEATURES             Location/Qualifiers
     source          1..4520
                     /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             152..172
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             182..520
                     /codon_start=1
                     /product="improved SpyCatcher variant that reacts with 
                     SpyTag003 at a rate approaching the diffusion limit (Keeble
                     et al., 2019)"
                     /label=SpyCatcher003
                     /translation="VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATME
                     LRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDG
                     EATEGDAHT"
     CDS             896..916
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1041..1058
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1111..1129)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1125..1172
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1209..1664
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1296..1315)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1506..1527
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1690..1794
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1795..1863,1864..2655)
                     /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(2013..2032)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2826..3414
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3315..3334
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3568..3585
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3600..3739
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3665..3684)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3825..3847
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3841..4032)
                     /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 tccccacaac tgagaacctt tatttccaag gtggggcgat
      181 ggttactact ctgagtggtc tgagcggcga acagggtccg tcgggtgaca tgacgacaga
      241 ggaagacagt gccacgcata ttaaattttc aaaacgtgat gaggacggtc gcgagttagc
      301 aggggcaact atggaattgc gtgactcctc cggcaaaacc atttctacat ggattagtga
      361 tgggcacgta aaggattttt atctgtaccc cggaaagtat acctttgtcg agacagcggc
      421 tcctgacggt tatgaagtgg caaccccgat cgaatttacg gtgaatgagg acgggcaggt
      481 gacggtagat ggggaagcca cagagggcga tgctcatacc gggtctggtg gatcgggagg
      541 ctctggcgta actactttaa gtgggttgag cggagaacaa gggcctagtg gagatatgac
      601 aaccgaggag gactccgcca cccatattaa atttagtaaa cgcgatgaag acggtcgcga
      661 gcttgcgggt gctactatgg agctgcgcga tagtagcggc aagaccatct cgacctggat
      721 ttcagatgga catgtcaaag acttttacct gtaccctggc aaatatacct ttgtggaaac
      781 ggcagctccg gatggatatg aggttgcgac gccgatcgag ttcacggtca acgaagatgg
      841 acaagttacg gtggacggtg aggcaactga aggagatgca ggtagttcag gctcagaaaa
      901 cttgtatttt cagggcggag gatcggggcg cggagtgccc cacattgtta tggtggccgc
      961 ctacaaacgc tataaataaa tggttgatgc ttgaggatcc gaattcgagc tccgtcgaca
     1021 agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
     1081 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     1141 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     1201 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1261 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     1321 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     1381 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     1441 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     1501 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     1561 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     1621 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     1681 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1741 ctcatgagac aataaccctg gtaaatgctt caataatatt gaaaaaggaa gagtatgagt
     1801 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     1861 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     1921 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     1981 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     2041 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     2101 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     2161 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     2221 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     2281 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     2341 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     2401 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     2461 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     2521 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     2581 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     2641 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     2701 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     2761 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2821 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2881 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2941 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     3001 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3061 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3121 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3181 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3241 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3301 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3361 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3421 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3481 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3541 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3601 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     3661 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     3721 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     3781 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     3841 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     3901 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagctt
     3961 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     4021 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     4081 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     4141 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     4201 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     4261 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     4321 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     4381 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     4441 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     4501 ccaggaccca acgctgcccg
//
