LOCUS       sequence-137281-        4115 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SpyCatcher with a peptide tag enabling 
            site-specific biotinylation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4115)
  AUTHORS   Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, 
            Howarth M
  TITLE     Programmable polyproteams built using twin peptide superglues.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 Jan 19. pii: 201519214.
  PUBMED    26787909
REFERENCE   2  (bases 1 to 4115)
  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. 2016 Jan 19. pii: 201519214."
COMMENT     Sequence Label: AviTag-SpyCatcher
FEATURES             Location/Qualifiers
     source          1..4115
                     /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             137..181
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             206..226
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             233..571
                     /codon_start=1
                     /product="truncated version of the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyCatcher
                     /note="forms a covalent isopeptide bond with the SpyTag 
                     peptide (Zakeri et al., 2012)"
                     /translation="VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATME
                     LRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     KATKGDAHI"
     CDS             636..653
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(706..724)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      720..767
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      804..1259
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(891..910)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1101..1122
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1285..1389
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1390..1458,1459..2250)
                     /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(1608..1627)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2421..3009
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2910..2929
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3163..3180
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3195..3334
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3260..3279)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3420..3442
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3436..3627)
                     /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 ggcagcggcc tgaacgatat ttttgaagcg cagaaaattg aatggcatga
      181 aggcgattac gacatcccaa cgaccgaaaa cctgtatttt cagggcgcca tggttgatac
      241 cttatcaggt ttatcaagtg agcaaggtca gtccggtgat atgacaattg aagaagatag
      301 tgctacccat attaaattct caaaacgtga tgaggacggc aaagagttag ctggtgcaac
      361 tatggagttg cgtgattcat ctggtaaaac tattagtaca tggatttcag atggacaagt
      421 gaaagatttc tacctgtatc caggaaaata tacatttgtc gaaaccgcag caccagacgg
      481 ttatgaggta gcaactgcta ttacctttac agttaatgag caaggtcagg ttactgtaaa
      541 tggcaaagca actaaaggtg acgctcatat ttaaatggtt gatgcttgag gatccgaatt
      601 cgagctccgt cgacaagctt gcgaccgcac tcgagcacca ccaccaccac cactgagatc
      661 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
      721 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
      781 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
      841 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
      901 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
      961 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1021 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1081 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1141 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1201 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     1261 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1321 tcaaatatgt atccgctcat gagacaataa ccctggtaaa tgcttcaata atattgaaaa
     1381 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1441 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     1501 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     1561 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     1621 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     1681 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     1741 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     1801 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     1861 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     1921 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     1981 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2041 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2101 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     2161 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     2221 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     2281 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2341 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2401 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2461 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2521 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     2581 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2641 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2701 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2761 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2821 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2881 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     2941 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3001 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3061 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3121 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3181 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3241 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     3301 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     3361 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3421 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     3481 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     3541 cagctcgttg agcttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     3601 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     3661 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     3721 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     3781 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     3841 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     3901 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     3961 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     4021 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     4081 catgcgcacc cgtggccagg acccaacgct gcccg
//
