LOCUS       sequence-313950-        6846 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human TLNRD1 F250D mutant in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6846)
  AUTHORS   Cowell AR, Jacquemet G, Singh AK, Varela L, Nylund AS, Ammon YC, 
            Brown DG, Akhmanova A, Ivaska J, Goult BT
  TITLE     Talin rod domain-containing protein 1 (TLNRD1) is a novel 
            actin-bundling protein which promotes filopodia formation.
  JOURNAL   J Cell Biol. 2021 Sep 6;220(9). pii: 212472. doi: 
            10.1083/jcb.202005214. Epub 2021 Jul 15.
  PUBMED    34264272
REFERENCE   2  (bases 1 to 6846)
  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: "J Cell 
            Biol."; date: "2021-09-6"; volume: "220(9). pii: 212472. doi"; 
            pages: " 10.1083/jcb.202005214. Epub 2021 Jul 15"
COMMENT     Sequence Label: human TLNRD1-F250D pET151
FEATURES             Location/Qualifiers
     source          1..6846
                     /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             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             110..151
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             152..172
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1339..1357)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1353..1400
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1638..1656)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1724..1828
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1829..1897,1898..2689)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2047..2066)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2860..3448
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3349..3368
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3602..3619
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3634..3773
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3699..3718)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3859..3881
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3875..4066)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5378..6460)
                     /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     6422..6441
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6461..6538)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6744..6763
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcatcatcac catcaccatg gtaagcctat
      121 ccctaaccct ctcctcggtc tcgattctac ggaaaacctg tattttcagg gaattgatcc
      181 cttcaccatg gcaagcggta gcgcaggtaa accgaccggt gaagcagcaa gtccggcacc
      241 ggcaagcgca attggtggtg caagcagcca gcctcgtaaa cgtctggtta gcgtttgtga
      301 tcattgcaaa ggtaaaatgc agctggttgc cgatctgctg ctgctgagca gcgaagcacg
      361 tccggttctg tttgaaggtc cggcaagcag cggtgccggt gcagaaagct ttgaacagtg
      421 tcgtgatacc attattgcac gtaccaaagg tctgagcatt ctgacccatg atgttcagag
      481 ccagctgaat atgggtcgtt ttggtgaagc cggtgatagc ctggttgaac tgggtgatct
      541 ggttgttagc ctgaccgaat gtagcgcaca tgcagcatat ctggcagcag ttgcaacacc
      601 gggtgcacag ccagcacagc ctggtctggt tgatcgttat cgtgttaccc gttgtcgtca
      661 tgaagttgaa cagggttgtg cagttctgcg tgccacaccg ctggcagata tgacaccgca
      721 gctgctgtta gaagttagcc agggtctgag ccgtaatctg aaatttctga ccgatgcatg
      781 tgcactggca agcgataaaa gccgtgatcg ttttagccgt gaacagttca aactgggtgt
      841 taaatgtatg agcaccagcg caagcgcact gctggcatgt gttcgtgaag ttaaagttgc
      901 accgagcgaa ctggcacgta gccgttgtgc actggatagc ggtccgctgg ttcaggcagt
      961 tagcgcactg gttggttttg caaccgaacc gcagtttctg ggtcgtgcag cagccgttag
     1021 cgcagaaggt aaagcagttc agaccgcaat tcttggtggc gcaatgagcg ttgttagcgc
     1081 ctgtgttctg ctgacccagt gtctgcgtga tctggcacag catcctgatg gtggtgccaa
     1141 aatgagcgat catcgtgaac gtctgcgtaa tagcgcatgt gcggttagcg aaggttgtac
     1201 cctgctgagc caggcactgc gtgaacgtag cagtccgcgt acactgcctc cggttaatag
     1261 caatagcgtg aattaaaagg gcgagctcag atccggctgc taacaaagcc cgaaaggaag
     1321 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1381 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatccc gcaagaggcc
     1441 cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat
     1501 gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg
     1561 tgataaacta ccgcattaaa gctagcttat cgatgataag ctgtcaaaca tgagaattaa
     1621 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     1681 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     1741 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     1801 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     1861 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     1921 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     1981 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     2041 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
     2101 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     2161 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     2221 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     2281 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     2341 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
     2401 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     2461 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     2521 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     2581 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     2641 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     2701 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     2761 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     2821 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     2881 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2941 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3001 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3061 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3121 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3181 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3241 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3301 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3361 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3421 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3481 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3541 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3601 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     3661 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     3721 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     3781 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3841 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3901 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     3961 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     4021 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     4081 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     4141 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     4201 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     4261 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     4321 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     4381 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     4441 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     4501 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc
     4561 gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt
     4621 gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt
     4681 tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
     4741 caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca
     4801 tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag
     4861 gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct
     4921 ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat
     4981 ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc
     5041 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     5101 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     5161 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     5221 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     5281 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     5341 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5401 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5461 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     5521 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     5581 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     5641 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     5701 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     5761 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     5821 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     5881 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     5941 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     6001 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     6061 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     6121 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     6181 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     6241 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     6301 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     6361 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     6421 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     6481 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     6541 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     6601 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     6661 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     6721 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     6781 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     6841 cgaaat
//
