LOCUS       sequence-204302-        5866 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5866)
  AUTHORS   Chinthalapudi K, Rangarajan ES, Brown DT, Izard T
  TITLE     Differential lipid binding of vinculin isoforms promotes 
            quasi-equivalent dimerization.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 Aug 23;113(34):9539-44. doi: 
            10.1073/pnas.1600702113. Epub 2016 Aug 8.
  PUBMED    27503891
REFERENCE   2  (bases 1 to 5866)
  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"; doi: 
            "10.1073/pnas.1600702113"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2016-08-23- 23"; volume: "113"; issue: "34"; pages: "9539-44"
COMMENT     Sequence Label: pET28-Metavinculin tail
FEATURES             Location/Qualifiers
     source          1..5866
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      7..595
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     496..515
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     749..766
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    781..920
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(846..865)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1006..1028
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1022..1213)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2022..3104)
                     /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     3066..3085
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3105..3182)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3388..3407
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3491..3510
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3491..3509
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3510..3534
                     /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             3549..3571
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3590..3613
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3614..3637
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             4221..4238
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4291..4309)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4305..4352
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4389..4844
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4476..4495)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4686..4707
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4936..5751)
                     /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(4936..5751)
                     /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     5659..5678
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5743..5762
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
       61 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
      121 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
      181 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
      241 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
      301 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
      361 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
      421 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
      481 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
      541 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
      601 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
      661 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
      721 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
      781 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
      841 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
      901 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
      961 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     1021 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     1081 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     1141 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     1201 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     1261 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     1321 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     1381 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     1441 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     1501 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     1561 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     1621 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     1681 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     1741 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     1801 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     1861 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     1921 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     1981 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     2041 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2101 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     2161 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     2221 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     2281 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     2341 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     2401 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     2461 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     2521 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     2581 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     2641 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     2701 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     2761 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     2821 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     2881 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     2941 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     3001 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     3061 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     3121 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     3181 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     3241 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     3301 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     3361 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     3421 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     3481 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     3541 gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca
     3601 tcatcaccat catctggaag ttctgttcca ggggccccat atgaatcagc cggtcaacca
     3661 gcccattctg gccgcggctc agtccttgca tcgggaagct accaagtggt ctagtaaggg
     3721 caatgacatc attgcagcag ccaagcgcat ggctctgctg atggctgaga tgtctcggct
     3781 ggtaagaggg ggcagtggta ccaagcgggc actcattcag tgtgccaagg acatcgccaa
     3841 ggcctcagat gaggtgactc ggttggccaa ggaggttgcc aagcagtgca cagataaacg
     3901 gattagaacc aacctcttac aggtatgtga gcgaatccca accataagca cccagctcaa
     3961 aatcctgtcc acagtgaagg ccaccatgct gggccggacc aacatcagtg atgaggagtc
     4021 tgagcaggcc acagagatgc tggttcacaa tgcccagaac ctcatgcagt ctgtgaagga
     4081 gactgtgcgg gaagctgaag ctgcttcaat caaaattcga acagatgctg gatttacact
     4141 gcgctgggtt agaaagactc cctggtacca gtgaggatcc gaattcgagc tccgtcgaca
     4201 agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
     4261 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     4321 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     4381 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4441 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4501 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4561 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     4621 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4681 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     4741 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     4801 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     4861 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4921 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     4981 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     5041 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     5101 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     5161 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     5221 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     5281 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     5341 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     5401 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     5461 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     5521 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     5581 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     5641 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     5701 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     5761 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     5821 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaagg
//
