LOCUS       sequence-324208-        6466 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of SpyCatcher with ddFLN4, ELP linker, His6x tag, and 
            ybbR Tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6466)
  AUTHORS   Yang B, Liu H, Liu Z, Doenen R, Nash MA
  TITLE     Influence of Fluorination on Single-Molecule Unfolding and Rupture 
            Pathways of a Mechanostable Protein Adhesion Complex.
  JOURNAL   Nano Lett. 2020 Nov 16. doi: 10.1021/acs.nanolett.0c04178.
  PUBMED    33191756
REFERENCE   2  (bases 1 to 6466)
  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: "Nano Lett. 
            2020 Nov 16. doi: 10.1021/acs.nanolett.0c04178."
COMMENT     Sequence Label: pET28a-ybbR-His-ELP(MV7E2)3-ddFLN4-SpyCatcher
FEATURES             Location/Qualifiers
     source          1..6466
                     /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"
     protein_bind    20..44
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             131..148
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1004..1342
                     /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"
     primer_bind     complement(1401..1419)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1415..1462
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1499..1954
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1586..1605)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1796..1817
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2046..2861)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2046..2861)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2769..2788
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2853..2872
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2983..3571
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3472..3491
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3725..3742
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3757..3896
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3822..3841)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3982..4004
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3998..4189)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4998..6080)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6042..6061
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6081..6158)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6364..6383
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gggtaccgac tctctggaat tcatcgcttc
      121 taaactggct catcatcatc accatcactg gggatccggc cacggcgtgg gtgttccagg
      181 tatgggtgtg cctggtgttg gtgttcctgg cgttggcgtg ccaggtgtgg gagtaccagg
      241 cgtaggcgtt cccggtgtag gcgtgcctgg cgtcggtgta cccggtgtcg gagtccctgg
      301 tgaaggtgtg ccaggcgaag gtgtgccggg cgtgggtgtt ccaggtatgg gtgtgcctgg
      361 tgttggtgtt cctggcgttg gcgtgccagg tgtgggagta ccaggcgtag gcgttcccgg
      421 tgtaggcgtg cctggcgtcg gtgtacccgg tgtcggagtc cctggtgaag gtgtgccagg
      481 cgaaggtgtg ccgggcgtgg gtgttccagg tatgggtgtg cctggtgttg gtgttcctgg
      541 cgttggcgtg ccaggtgtgg gagtaccagg cgtaggcgtt cccggtgtag gcgtgcctgg
      601 cgtcggtgta cccggtgtcg gagtccctgg tgaaggtgtg ccaggcgaag gtgtgccggg
      661 ctggccgtca ggttctgctg acccggaaaa atcttacgct gaaggtccgg gtctggacgg
      721 tggtgaatgc ttccagccgt ctaaattcaa aatccacgct gttgacccgg acggtgttca
      781 ccgtaccgac ggtggtgacg gtttcgttgt taccatcgaa ggtccggctc cggttgaccc
      841 ggttatggtt gacaacggtg acggtaccta cgacgttgaa ttcgaaccga aagaagctgg
      901 tgactacgtt atcaacctga ccctggacgg tgacaacgtt aacggtttcc cgaaaaccgt
      961 taccgttaaa ccggctccgg gctccggttc tggttccggt agcgttgata ccctgagcgg
     1021 tctgagcagc gaacagggtc agagcggtga tatgaccatt gaagaagata gcgcaaccca
     1081 catcaaattc agcaaacgtg atgaagatgg caaagaactg gcaggcgcaa ccatggaact
     1141 gcgtgatagc agcggtaaaa ccattagcac ctggattagt gatggtcagg tgaaagattt
     1201 ttatctgtat ccgggtaaat ataccttcgt tgaaaccgca gcaccggatg gttatgaagt
     1261 tgcaaccgca attaccttta ccgtgaatga acaaggtcag gttaccgtta atggtaaagc
     1321 aaccaaaggt gatgcccaca tctaactcga ggatccggct gctaacaaag cccgaaagga
     1381 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     1441 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     1501 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1561 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1621 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1681 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     1741 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     1801 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     1861 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     1921 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     1981 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     2041 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     2101 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2161 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2221 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2281 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     2341 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     2401 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     2461 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     2521 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     2581 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     2641 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     2701 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     2761 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     2821 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     2881 tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt
     2941 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3001 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3061 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3121 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3181 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3241 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3301 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3361 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3421 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3481 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3541 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3601 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3661 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3721 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3781 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     3841 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     3901 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3961 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4021 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     4081 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     4141 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     4201 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     4261 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     4321 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     4381 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     4441 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     4501 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     4561 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     4621 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     4681 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     4741 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     4801 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     4861 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     4921 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     4981 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5041 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     5101 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     5161 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     5221 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     5281 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     5341 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     5401 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     5461 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     5521 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     5581 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     5641 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     5701 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     5761 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     5821 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     5881 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     5941 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     6001 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     6061 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     6121 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     6181 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     6241 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     6301 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     6361 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     6421 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaat
//
