LOCUS       sequence-200985-        4130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SpyCatcher002 with a peptide tag enabling 
            site-specific biotinylation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4130)
  AUTHORS   Keeble AH, Banerjee A, Reddington SC, Ferla MP, Howarth M, Khairil 
            Anuar INA
  TITLE     Evolving accelerated amidation by SpyTag/SpyCatcher to analyze 
            membrane dynamics.
  JOURNAL   Angew Chem Int Ed Engl. 2017 Oct 10. doi: 10.1002/anie.201707623.
  PUBMED    29024296
REFERENCE   2  (bases 1 to 4130)
  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: "Angew Chem 
            Int Ed Engl. 2017 Oct 10. doi: 10.1002/anie.201707623."
COMMENT     Sequence Label: pDEST14-Avitag-SpyCatcher002
FEATURES             Location/Qualifiers
     source          1..4130
                     /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             117..161
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             186..206
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             213..551
                     /codon_start=1
                     /product="improved SpyCatcher variant that shows rapid 
                     reaction with SpyTag002 (Keeble et al., 2017)"
                     /label=SpyCatcher002
                     /translation="VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATME
                     LRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     EATKGDAHT"
     CDS             631..648
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(701..719)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      715..762
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      799..1254
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(886..905)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1096..1117
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1280..1384
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1385..1453,1454..2245)
                     /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(1603..1622)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2416..3004
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2905..2924
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3158..3175
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3190..3329
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3255..3274)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3415..3437
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3431..3622)
                     /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 ggcagcggcc
      121 tgaacgatat ttttgaagcg cagaaaattg aatggcatga aggcgattac gacatcccaa
      181 cgaccgaaaa cctgtatttt cagggcgcca tggtaaccac cttatcaggt ttatcaggtg
      241 agcaaggtcc gtccggtgat atgacaactg aagaagatag tgctacccat attaaattct
      301 caaaacgtga tgaggacggc cgtgagttag ctggtgcaac tatggagttg cgtgattcat
      361 ctggtaaaac tattagtaca tggatttcag atggacatgt gaaggatttc tacctgtatc
      421 caggaaaata tacatttgtc gaaaccgcag caccagacgg ttatgaggta gcaactgcta
      481 ttacctttac agttaatgag caaggtcagg ttactgtaaa tggcgaagca actaaaggtg
      541 acgctcatac tggatccagt ggtagctaaa tggttgatgc ttgaggatcc gaattcgagc
      601 tccgtcgaca agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct
      661 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
      721 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
      781 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
      841 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
      901 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
      961 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     1021 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     1081 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     1141 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     1201 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     1261 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1321 tatgtatccg ctcatgagac aataaccctg gtaaatgctt caataatatt gaaaaaggaa
     1381 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     1441 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     1501 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     1561 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     1621 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     1681 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     1741 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     1801 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     1861 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     1921 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     1981 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     2041 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     2101 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     2161 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     2221 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     2281 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     2341 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2401 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2461 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2521 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     2581 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2641 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2701 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     2761 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2821 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2881 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2941 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3001 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3061 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3121 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3181 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     3241 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     3301 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     3361 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3421 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     3481 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     3541 cgttgagctt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     3601 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     3661 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     3721 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     3781 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     3841 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     3901 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     3961 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     4021 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     4081 gcacccgtgg ccaggaccca acgctgcccg agatctcgat cccgcgaaat
//
