LOCUS       sequence-311039-        4076 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyCatcher003 S49C in bacterial cytoplasm, with a unique 
            cysteine for chemical coupling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4076)
  AUTHORS   Keeble AH, Turkki P, Stokes S, Khairil Anuar INA, Rahikainen R, 
            Hytonen VP, Howarth M
  TITLE     Approaching infinite affinity through engineering of peptide-protein
            interaction.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Dec 10. pii: 1909653116. doi: 
            10.1073/pnas.1909653116.
  PUBMED    31822621
REFERENCE   2  (bases 1 to 4076)
  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: "Proc Natl 
            Acad Sci U S A. 2019 Dec 10. pii: 1909653116. doi: 
            10.1073/pnas.1909653116."
COMMENT     Sequence Label: SpyCatcher003 S49C
FEATURES             Location/Qualifiers
     source          1..4076
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             51..73
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             93..110
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             132..152
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             159..497
                     /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
                     LRDCSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDG
                     EATEGDAHT"
     CDS             577..594
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(647..665)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      661..708
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      745..1200
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(832..851)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1042..1063
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1226..1330
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1331..1399,1400..2191)
                     /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(1549..1568)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2362..2950
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2851..2870
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3104..3121
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3136..3275
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3201..3220)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3361..3383
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3377..3568)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac
       61 tttaagaagg agatatacat atgtcgtact accatcacca tcaccatcac gattacgaca
      121 tcccaacgac cgaaaacctg tattttcagg gcgccatggt aaccacctta tcaggtttat
      181 caggtgagca aggtccgtcc ggtgatatga caactgaaga agatagtgct acccatatta
      241 aattctcaaa acgtgatgag gacggccgtg agttagctgg tgcaactatg gagttgcgtg
      301 attgctctgg taaaactatt agtacatgga tttcagatgg acatgtgaag gatttctacc
      361 tgtatccagg aaaatataca tttgtcgaaa ccgcagcacc agacggttat gaggtagcaa
      421 ctccaattga atttacagtt aatgaggacg gtcaggttac tgtagatggt gaagcaactg
      481 aaggtgacgc tcatactgga tccagtggta gctaaatggt tgatgcttga ggatccgaat
      541 tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat
      601 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
      661 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
      721 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
      781 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
      841 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
      901 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
      961 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1021 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1081 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     1141 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     1201 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1261 ttcaaatatg tatccgctca tgagacaata accctggtaa atgcttcaat aatattgaaa
     1321 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     1381 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     1441 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     1501 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     1561 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     1621 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     1681 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     1741 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     1801 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     1861 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     1921 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     1981 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     2041 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     2101 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     2161 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     2221 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     2281 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2341 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2401 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2461 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     2521 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2581 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2641 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     2701 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2761 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     2821 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     2881 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     2941 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3001 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3061 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3121 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3181 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     3241 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     3301 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     3361 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     3421 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     3481 ccagctcgtt gagcttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     3541 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     3601 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     3661 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     3721 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     3781 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     3841 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     3901 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     3961 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4021 tcatgcgcac ccgtggccag gacccaacgc tgcccgagat ctcgatcccg cgaaat
//
