LOCUS       sequence-330252-        5644 bp    DNA     circular SYN 14-APR-2025
DEFINITION  For expression of human talin-1 head (residues 1-405) 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 5644)
  AUTHORS   Kukkurainen S, Azizi L, Zhang P, Jacquier MC, Baikoghli M, von Essen
            M, Tuukkanen A, Laitaoja M, Liu X, Rahikainen R, Orlowski A, Janis 
            J, Maatta JAE, Varjosalo M, Vattulainen I, Rog T, Svergun D, Cheng 
            RH, Wu J, Hytonen VP, Wehrle-Haller B
  TITLE     The F1 loop of the talin head domain acts as a gatekeeper in 
            integrin activation and clustering.
  JOURNAL   J Cell Sci. 2020 Oct 12;133(19). pii: 133/19/jcs239202. doi: 
            10.1242/jcs.239202.
  PUBMED    33046605
REFERENCE   2  (bases 1 to 5644)
  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"; journalName: "J Cell 
            Sci."; date: "2020-10-12"; volume: "133(19). pii: 133/19/jcs239202. 
            doi"; pages: " 10.1242/jcs.239202"
COMMENT     Sequence Label: t1-405
FEATURES             Location/Qualifiers
     source          1..5644
                     /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(1767..1785)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1768..1785)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1828..1845)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1998..2084
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2176..2203
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2222..2313
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2314..2382,2383..3174)
                     /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(2532..2551)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3345..3933
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3834..3853
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4087..4104
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4119..4259
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4185..4204)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4345..4367
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4445..4522
                     /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             4523..5605
                     /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(4542..4561)
                     /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 aagctaatga gtctagaggg ccctattcta tagtgtcacc taaatgctaa agcttggctg
     1801 ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg
     1861 tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc
     1921 gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt
     1981 agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     2041 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
     2101 tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca
     2161 ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc
     2221 ttttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2281 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2341 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2401 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2461 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2521 tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc
     2581 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2641 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2701 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2761 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2821 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     2881 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2941 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3001 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     3061 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3121 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3181 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3241 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3301 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3361 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3421 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3481 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3541 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3601 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3661 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3721 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3781 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3841 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3901 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3961 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4021 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4081 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     4141 acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat
     4201 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4261 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4321 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4381 caccgaaacg cgcgaggcag cagatcaatt cgcgcgcgaa ggcgaagcgg catgcattta
     4441 cgttgacacc atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     4501 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     4561 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     4621 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     4681 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     4741 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     4801 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     4861 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     4921 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     4981 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     5041 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     5101 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     5161 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     5221 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     5281 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     5341 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     5401 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     5461 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     5521 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     5581 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag cgcgaattga
     5641 tctg
//
