LOCUS       sequence-324211-        6736 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Coh (type III from Ruminococcus flavefaciens) 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 6736)
  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 6736)
  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-Coh-ddFLN4-ELP(MV7E2)3-His-ybbR
FEATURES             Location/Qualifiers
     source          1..6736
                     /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             1553..1570
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1671..1689)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1685..1732
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1769..2224
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1856..1875)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2066..2087
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2316..3131)
                     /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(2316..3131)
                     /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     3039..3058
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3123..3142
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3253..3841
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3742..3761
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3995..4012
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4027..4166
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4092..4111)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4252..4274
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4268..4459)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5268..6350)
                     /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     6312..6331
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6351..6428)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6634..6653
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taagaaggag atatacatat ggcgctcaca gacagaggaa tgacttacga
      121 tctcgatcct aaggatggat catctgctgc tacaaagcca gttctcgaag taactaagaa
      181 ggttttcgac acagctgctg atgctgctgg ccagactgtt acagttgaat tcaaggttag
      241 cggtgcagaa ggcaagtacg ctacaactgg ttatcacatc tactgggatg agagacttga
      301 agttgttgca accaagacag gtgcatacgc taagaagggc gcagctcttg aagattcaag
      361 cttagctaag gctgagaaca acggtaacgg tgttttcgtt gcttcaggtg ctgatgatga
      421 ttttggtgct gacggtgtaa tgtggactgt tgaactcaag gttccagcag acgctaaggc
      481 tggcgatgta tatccaatcg acgttgctta tcagtgggat ccttcaaagg gtgacctttt
      541 cacagataac aaggactcag cacagggcaa gctgatgcag gcttacttct tcacacaggg
      601 tatcaagtca tcttctaatc cttcaacaga tgagtacctc gtaaaggcta acgctacata
      661 tgctgatggt tacattgcaa tcaaggctgg tgagccaggc tccggttctg gatccggttc
      721 tgctgacccg gaaaaatctt acgctgaagg tccgggtctg gacggtggtg aatgcttcca
      781 gccgtctaaa ttcaaaatcc acgctgttga cccggacggt gttcaccgta ccgacggtgg
      841 tgacggtttc gttgttacca tcgaaggtcc ggctccggtt gacccggtta tggttgacaa
      901 cggtgacggt acctacgacg ttgaattcga accgaaagaa gctggtgact acgttatcaa
      961 cctgaccctg gacggtgaca acgttaacgg tttcccgaaa accgttaccg ttaaaccggc
     1021 tccgggctcc ggttctggtt cccacggcgt gggtgttcca ggtatgggtg tgcctggtgt
     1081 tggtgttcct ggcgttggcg tgccaggtgt gggagtacca ggcgtaggcg ttcccggtgt
     1141 aggcgtgcct ggcgtcggtg tacccggtgt cggagtccct ggtgaaggtg tgccaggcga
     1201 aggtgtgccg ggcgtgggtg ttccaggtat gggtgtgcct ggtgttggtg ttcctggcgt
     1261 tggcgtgcca ggtgtgggag taccaggcgt aggcgttccc ggtgtaggcg tgcctggcgt
     1321 cggtgtaccc ggtgtcggag tccctggtga aggtgtgcca ggcgaaggtg tgccgggcgt
     1381 gggtgttcca ggtatgggtg tgcctggtgt tggtgttcct ggcgttggcg tgccaggtgt
     1441 gggagtacca ggcgtaggcg ttcccggtgt aggcgtgcct ggcgtcggtg tacccggtgt
     1501 cggagtccct ggtgaaggtg tgccaggcga aggtgtgccg ggctggcgcg gtcatcatca
     1561 tcaccatcat ggtagcgact ctctggaatt catcgcttct aaactggctt aactcgagta
     1621 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     1681 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     1741 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     1801 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1861 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1921 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1981 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2041 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2101 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2161 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     2221 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2281 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     2341 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     2401 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     2461 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     2521 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     2581 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     2641 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     2701 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     2761 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     2821 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     2881 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     2941 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     3001 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     3061 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     3121 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     3181 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3241 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3301 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3361 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     3421 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3481 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3541 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3601 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3661 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3721 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3781 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3841 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3901 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3961 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     4021 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     4081 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     4141 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     4201 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4261 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     4321 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     4381 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     4441 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     4501 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     4561 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     4621 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     4681 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     4741 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     4801 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     4861 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     4921 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     4981 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     5041 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     5101 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     5161 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     5221 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     5281 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5341 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     5401 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     5461 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     5521 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     5581 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     5641 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     5701 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     5761 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     5821 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     5881 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     5941 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     6001 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     6061 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     6121 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     6181 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     6241 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     6301 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     6361 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     6421 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     6481 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     6541 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     6601 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     6661 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     6721 ctcgatcccg cgaaat
//
