LOCUS       sequence-462964-        5589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Avi-CC-S2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5589)
  AUTHORS   Liu S, Liu J, Foote A, Ogasawara H, Al Abdullatif S, Batista VS, 
            Salaita K
  TITLE     Digital and Tunable Genetically Encoded Tension Sensors Based on 
            Engineered Coiled-Coils.
  JOURNAL   Angew Chem Int Ed Engl. 2025 Jan 13:e202407359. doi: 
            10.1002/anie.202407359.
  PUBMED    39805005
REFERENCE   2  (bases 1 to 5589)
  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. 2025 Jan 13:e202407359. doi: 10.1002/anie.202407359."
COMMENT     Sequence Label: pET28a-AviTag-CC-S2
FEATURES             Location/Qualifiers
     source          1..5589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     950..969
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(989..1066)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1272..1291
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1375..1394
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1375..1393
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1394..1418
                     /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             1433..1455
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1474..1491
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1501..1518
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1525..1569
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1642..1653
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             1828..1845
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1898..1916)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1912..1959
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1996..2451
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2083..2102)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2293..2314
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2543..3358)
                     /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(2543..3358)
                     /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     3266..3285
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3350..3369
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3480..4068
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3969..3988
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4222..4239
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4254..4393
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4319..4338)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4479..4501
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4495..4686)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5495..5589,1..988))
                     /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"
ORIGIN
        1 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
       61 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
      121 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
      181 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
      241 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
      301 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
      361 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
      421 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
      481 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
      541 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
      601 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
      661 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
      721 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
      781 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
      841 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
      901 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
      961 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     1021 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     1081 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     1141 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     1201 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     1261 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     1321 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
     1381 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
     1441 actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca cagcagcggc
     1501 ctggtgccgc gcggcagcca tatgggtctg aatgatattt tcgaagcgca gaaaattgaa
     1561 tggcatgaag gtagcggagg cggaagcgca cagcttgaga aggaattgca ggccctggag
     1621 aaagaaaacg cccaactgga gtgggagtta caagcctccg agaaagagtc ggcgcaaggc
     1681 ggttcgggtg gctcacaggc gtccaaaaag aagtccgctc agcttaaatg gaaactgcaa
     1741 gcaaataaga agaagttggc gcagttgaag aaaaaattac aagcaggcgg aagttcgagc
     1801 tgttaaaagc ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct
     1861 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     1921 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     1981 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2041 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2101 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2161 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2221 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2281 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2341 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2401 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     2461 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2521 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     2581 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     2641 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     2701 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     2761 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     2821 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     2881 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     2941 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     3001 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     3061 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     3121 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     3181 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     3241 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     3301 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     3361 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     3421 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3481 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3541 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3601 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     3661 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3721 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3781 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3841 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3901 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3961 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4021 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4081 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4141 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4201 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     4261 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     4321 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     4381 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4441 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4501 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     4561 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     4621 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     4681 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     4741 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     4801 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     4861 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     4921 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     4981 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     5041 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     5101 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     5161 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     5221 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     5281 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     5341 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     5401 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     5461 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5521 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5581 tgggcgcca
//
