LOCUS       sequence-433331-        4607 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses DoubleCatcher H-lock in bacterial cells. DoubleCatcher 
            H-lock is a variant of DoubleCatcher in which each SpyCatcher003 
            moiety is separated by an alpha helical linker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4607)
  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 4607)
  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-H-Lock
FEATURES             Location/Qualifiers
     source          1..4607
                     /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             983..1003
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1128..1145
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1198..1216)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1212..1259
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1296..1751
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1383..1402)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1593..1614
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1777..1881
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1882..1950,1951..2742)
                     /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(2100..2119)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2913..3501
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3402..3421
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3655..3672
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3687..3826
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3752..3771)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3912..3934
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3928..4119)
                     /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 gggtctcccg ccaacctgaa
      541 ggctttggag gcccagaagc agaaggagca gagacaggcc gccgaggagc tggccaacgc
      601 caagaaactg aaagagcagc tggagaaggg atccgtaact actttaagtg ggttgagcgg
      661 agaacaaggg cctagtggag atatgacaac cgaggaggac tccgccaccc atattaaatt
      721 tagtaaacgc gatgaagacg gtcgcgagct tgcgggtgct actatggagc tgcgcgatag
      781 tagcggcaag accatctcga cctggatttc agatggacat gtcaaagact tttacctgta
      841 ccctggcaaa tatacctttg tggaaacggc agctccggat ggatatgagg ttgcgacgcc
      901 gatcgagttc acggtcaacg aagatggaca agttacggtg gacggtgagg caactgaagg
      961 agatgcaggt agttcaggct cagaaaactt gtattttcag ggcggaggat cggggcgcgg
     1021 agtgccccac attgttatgg tggccgccta caaacgctat aaataaatgg ttgatgcttg
     1081 aggatccgaa ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac caccaccacc
     1141 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     1201 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     1261 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
     1321 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     1381 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     1441 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     1501 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     1561 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     1621 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     1681 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     1741 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     1801 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctggta aatgcttcaa
     1861 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     1921 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     1981 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     2041 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     2101 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     2161 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     2221 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     2281 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     2341 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     2401 gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact
     2461 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     2521 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     2581 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     2641 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     2701 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     2761 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     2821 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2881 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2941 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3001 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     3061 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3121 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3181 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3241 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3301 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3361 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3421 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3481 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3541 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3601 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3661 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     3721 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3781 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     3841 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3901 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3961 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     4021 ttcatccgcg tccagctcgt tgagcttctc cagaagcgtt aatgtctggc ttctgataaa
     4081 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     4141 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     4201 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     4261 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     4321 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     4381 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     4441 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     4501 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     4561 caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccg
//
