LOCUS       sequence-324200-        5450 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Coh (type III from Ruminococcus flavefaciens) with 
            His6x tag and SpyTag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5450)
  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 5450)
  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: pQE80L-Coh-His-SpyTag
FEATURES             Location/Qualifiers
     source          1..5450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..15,16..21,22..38,39..45)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    21..37
                     /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)."
     protein_bind    53..69
                     /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             88..99
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             121..138
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             763..780
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             796..834
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     terminator      898..992
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1036..1695
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1102..1121)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1760..1846
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1939..3021)
                     /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     2983..3002
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3022..3099)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(3183..3205)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3291..3431
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3346..3365
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3446..3463)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3617..4205)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3697..3716)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4376..5167,5168..5236))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4999..5018
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5237..5341)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5409..5427
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcataaaaaa tttatttgct ttgtgagcgg ataacaatta taatagattc aattgtgagc
       61 ggataacaat ttcacacaga attcattaaa gaggagaaat taactatgaa aggcagcagc
      121 catcatcatc atcatcacgt cgacgcccat attgtcgcgc tcacagacag aggaatgact
      181 tacgatctcg atcctaagga tggatcatct gctgctacaa agccagttct cgaagtaact
      241 aagaaggttt tcgacacagc tgctgatgct gctggccaga ctgttacagt tgaattcaag
      301 gttagcggtg cagaaggcaa gtacgctaca actggttatc acatctactg ggatgagaga
      361 cttgaagttg ttgcaaccaa gacaggtgca tacgctaaga agggcgcagc tcttgaagat
      421 tcaagcttag ctaaggctga gaacaacggt aacggtgttt tcgttgcttc aggtgctgat
      481 gatgattttg gtgctgacgg tgtaatgtgg actgttgaac tcaaggttcc agcagacgct
      541 aaggctggcg atgtatatcc aatcgacgtt gcttatcagt gggatccttc aaagggtgac
      601 cttttcacag ataacaagga ctcagcacag ggcaagctga tgcaggctta cttcttcaca
      661 cagggtatca agtcatcttc taatccttca acagatgagt acctcgtaaa ggctaacgct
      721 acatatgctg atggttacat tgcaatcaag gctggtgagc cacatcacca tcaccatcac
      781 ggcgggctgc tcgacgccca tattgtcatg gttgatgcat acaagccgac gaagctcgag
      841 tggaagaaat aaggatccgg ctgctaacaa agcccgaagc ttaattagct gagcttggac
      901 tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc agaacgctcg
      961 gttgccgccg ggcgtttttt attggtgaga atccaagcca gcttggcgag attttcagga
     1021 gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa
     1081 tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag
     1141 accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt
     1201 tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga atttcgtatg
     1261 gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc
     1321 catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag
     1381 tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct
     1441 aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt
     1501 tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa
     1561 tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt
     1621 tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg
     1681 cagggcgggg cgtaattttt ttaaggcagt tattggtgcc cttaaacgcc tggggtaatg
     1741 actctctagc ttgaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     1801 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cctctagatt
     1861 acgtgcagtc gatgataagc tgtcaaacat gagaattgtg cctaatgagt gagctaactt
     1921 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     1981 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     2041 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     2101 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     2161 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     2221 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     2281 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     2341 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     2401 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     2461 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     2521 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     2581 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     2641 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     2701 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     2761 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     2821 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     2881 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     2941 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     3001 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     3061 tgccataccg cgaaaggttt tgcaccattc gatggtgtcg gaatttcggg cagcgttggg
     3121 tcctggccac gggtgcgcat gatctagagc tgcctcgcgc gtttcggtga tgacggtgaa
     3181 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     3241 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     3301 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     3361 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     3421 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     3481 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     3541 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     3601 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     3661 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3721 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     3781 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     3841 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     3901 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     3961 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4021 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     4081 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4141 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4201 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4261 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4321 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     4381 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     4441 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     4501 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     4561 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     4621 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     4681 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     4741 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     4801 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     4861 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     4921 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     4981 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     5041 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     5101 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     5161 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     5221 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     5281 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     5341 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     5401 cctataaaaa taggcgtatc acgaggccct ttcgtcttca ccttgagaaa
//
