LOCUS       sequence-330255-        5734 bp    DNA     circular SYN 14-APR-2025
DEFINITION  For expression of human talin head (residues 1-435) in E. coli. 
            N-terminal His6-tag, Xpress-epitope (DLYDDDDK) and enterokinase 
            cleavage site for tag removal..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5734)
  AUTHORS   Zhang P, Azizi L, Kukkurainen S, Gao T, Baikoghli M, Jacquier MC, 
            Sun Y, Maatta JAE, Cheng RH, Wehrle-Haller B, Hytonen VP, Wu J
  TITLE     Crystal structure of the FERM-folded talin head reveals the 
            determinants for integrin binding.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Dec 22;117(51):32402-32412. doi: 
            10.1073/pnas.2014583117. Epub 2020 Dec 7.
  PUBMED    33288722
REFERENCE   2  (bases 1 to 5734)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1073/pnas.2014583117"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2020-12-22- 22"; volume: "117"; issue: "51"; pages: 
            "32402-32412"
COMMENT     Sequence Label: t1-435
FEATURES             Location/Qualifiers
     source          1..5734
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(193..198,199..215,216..222)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    230..246
                     /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)."
     primer_bind     235..257
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    263..332
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             383..409
                     /codon_start=1
                     /product="synthetic cistron containing a ribosome binding 
                     site (Shine-Dalgarno sequence), for enhancing the bacterial
                     expression of a downstream cistron (Schoner, 1997)"
                     /label=minicistron
                     /note="This first cistron should terminate 3 bp upstream of
                     the ATG for the second cistron."
                     /translation="MYRLNKEE"
     CDS             425..442
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             446..478
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(482..490,491..505)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     promoter        complement(1857..1875)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1858..1875)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1918..1935)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2088..2174
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2266..2293
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2312..2403
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2404..2472,2473..3264)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2622..2641)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3435..4023
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3924..3943
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4177..4194
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4209..4349
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4275..4294)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4435..4457
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4535..4612
                     /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."
     CDS             4613..5695
                     /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     complement(4632..4651)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc
       61 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc
      121 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc
      181 tgaaatgagc tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgtgagcgga
      241 taacaatttc acacaggaaa cagcgccgct gagaaaaagc gaagcggcac tgctctttaa
      301 caatttatca gacaatctgt gtgggcactc gaccggaatt atcgattaac tttattatta
      361 aaaattaaag aggtatatat taatgtatcg attaaataag gaggaataaa ccatgggggg
      421 ttctcatcat catcatcatc atggtatggc tagcatgact ggtggacagc aaatgggtcg
      481 ggatctgtac gacgatgacg ataaggatcg atggatccga cctcgagcca tggttgcact
      541 ttcactgaag atcagcattg ggaatgtggt gaagacgatg cagtttgagc cgtctaccat
      601 ggtgtacgac gcctgccgca tcattcgtga gcggatccca gaggccccag ctggtcctcc
      661 cagcgacttt gggctctttc tgtcagatga tgaccccaaa aagggtatat ggctggaggc
      721 tgggaaagct ttggactact acatgctccg aaatggggac actatggagt acaggaagaa
      781 acagagaccc ctgaagatcc gtatgctgga tggaactgtg aagacgatca tggtggatga
      841 ctctaagact gtcactgaca tgctcatgac catctgtgcc cgcattggca tcaccaatca
      901 tgatgaatat tcattggttc gagagctgat ggaagagaaa aaggaggaaa taacagggac
      961 cttaagaaag gacaagacat tgctgcgaga tgaaaagaag atggagaaac taaagcagaa
     1021 attgcacaca gatgatgagt tgaactggct ggaccatggt cggacactga gggagcaggg
     1081 tgtagaggag cacgagacgc tgctgctgcg gaggaagttc ttttactcag accagaatgt
     1141 ggattcccgg gaccctgtac agctgaacct cctgtatgtg caggcacgag atgacatcct
     1201 gaatggctcc caccctgtct cctttgacaa ggcctgtgag tttgctggct tccaatgcca
     1261 gatccagttt gggccccaca atgagcagaa gcacaaggct ggcttccttg acctgaagga
     1321 cttcctgccc aaggagtatg tgaagcagaa gggagagcgt aagatcttcc aggcacacaa
     1381 gaattgtggg cagatgagtg agattgaggc caaggtccgc tacgtgaagc tagcccgttc
     1441 tctcaagact tacggtgtct ccttcttcct ggtgaaggaa aaaatgaaag ggaagaacaa
     1501 gctagtgccc aggcttctgg gcatcaccaa ggagtgtgtg atgcgagtgg atgagaagac
     1561 caaggaagtg atccaggagt ggaacctcac caacatcaaa cgctgggctg cgtctcccaa
     1621 aagcttcacc ctggattttg gagattacca agatggctat tactcagtac agacaactga
     1681 aggggagcag attgcacagc tcattgccgg ctacatcgat atcatcctga agaagaaaaa
     1741 aagcaaggat cactttgggc tggaaggaga tgaggagtct actatgctgg aggactcagt
     1801 gtcccccaaa aagtcaacag tcctgcagca gcaataatga gtctagaggg ccctattcta
     1861 tagtgtcacc taaatgctaa agcttggctg ttttggcgga tgagagaaga ttttcagcct
     1921 gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag
     1981 tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga
     2041 tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa
     2101 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
     2161 tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt
     2221 ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga
     2281 cggatggcct ttttgcgttt ctacaaactc ttttgtttat ttttctaaat acattcaaat
     2341 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2401 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2461 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     2521 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     2581 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     2641 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     2701 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     2761 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     2821 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     2881 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     2941 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3001 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3061 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     3121 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     3181 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3241 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     3301 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3361 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3421 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3481 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3541 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     3601 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3661 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3721 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3781 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3841 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3901 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3961 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4021 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4081 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     4141 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     4201 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4261 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     4321 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     4381 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4441 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag cagatcaatt
     4501 cgcgcgcgaa ggcgaagcgg catgcattta cgttgacacc atcgaatggt gcaaaacctt
     4561 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     4621 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     4681 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     4741 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     4801 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     4861 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     4921 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     4981 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     5041 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     5101 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     5161 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     5221 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     5281 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     5341 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     5401 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     5461 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     5521 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     5581 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     5641 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     5701 acgcaattaa tgtaagttag cgcgaattga tctg
//
