LOCUS       sequence-462233-        5588 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expresses DLC1 binding peptide to Talin R8 domain in chimera
            with meGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5588)
  AUTHORS   Dahal N, Sharma S, Phan B, Eis A, Popa I
  TITLE     Mechanical regulation of talin through binding and history-dependent
            unfolding.
  JOURNAL   Sci Adv. 2022 Jul 15;8(28):eabl7719. doi: 10.1126/sciadv.abl7719. 
            Epub 2022 Jul 15.
  PUBMED    35857491
REFERENCE   2  (bases 1 to 5588)
  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: "Sci Adv."; 
            date: "2022-07-15"; volume: "8(28):eabl7719. doi"; pages: " 
            10.1126/sciadv.abl7719. Epub 2022 Jul 15"
COMMENT     Sequence Label: pQE-meGFP-DLC1
FEATURES             Location/Qualifiers
     source          1..5588
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     164..183
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     423..444
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(493..510)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             529..540
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    559..575
                     /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)."
     promoter        complement(join(583..589,590..606,607..612,613..627))
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    591..607
                     /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     complement(651..669)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        737..841
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(842..910,911..1702)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1060..1079)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1873..2461
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2362..2381
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2615..2632
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2647..2787
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2713..2732)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2873..2895
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        2979..3056
                     /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             3057..4139
                     /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(3076..3095)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     terminator      complement(4232..4318)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4383..5042)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4957..4976
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(5086..5180)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(5361..5588,1..483,484..486))
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(5403..5424)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 cttgatgccg ttcttctgct tgtcggccat gatatagacg ttgtggctgt tgtagttgta
       61 ctccagcttg tgccccagga tgttgccgtc ctccttgaag tcgatgccct tcagctcgat
      121 gcggttcacc agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc
      181 gtccttgaag aagatggtgc gctcctggac gtagccttcg ggcatggcgg acttgaagaa
      241 gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt aggtcagggt
      301 ggtcacgagg gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt
      361 cagcttgccg taggtggcat cgccctcgcc ctcgccggac acgctgaact tgtggccgtt
      421 tacgtcgccg tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt
      481 gctcacggat ccgtgatggt gatggtgatg cgatcctctc atagttaatt tctcctcttt
      541 aatgaattct gtgtgaaatt gttatccgct cacaattgaa tctattataa ttgttatccg
      601 ctcacaaagc aaataaattt tttatgattt ctcgaggtga agacgaaagg gcctcgtgat
      661 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
      721 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
      781 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
      841 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
      901 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
      961 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     1021 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     1081 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     1141 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     1201 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     1261 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     1321 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     1381 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     1441 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     1501 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     1561 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     1621 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     1681 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     1741 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     1801 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     1861 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     1921 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     1981 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     2041 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2101 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2161 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     2221 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     2281 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     2341 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     2401 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     2461 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     2521 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     2581 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     2641 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     2701 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     2761 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     2821 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     2881 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct ctagatcatg
     2941 cgcacccgtg gccaggaccc aacgctgccc gaaattccga caccatcgaa tggtgcaaaa
     3001 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     3061 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     3121 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     3181 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     3241 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     3301 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     3361 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     3421 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     3481 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     3541 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     3601 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     3661 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     3721 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     3781 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     3841 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     3901 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     3961 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     4021 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     4081 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     4141 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac aattctcatg tttgacagct
     4201 tatcatcgac tgcacgtaat ctagagggcg gatttgtcct actcaggaga gcgttcaccg
     4261 acaaacaaca gataaaacga aaggcccagt ctttcgactg agcctttcgt tttatttgat
     4321 gcctcaagct agagagtcat taccccaggc gtttaagggc accaataact gccttaaaaa
     4381 aattacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg
     4441 acatggaagc catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg
     4501 tcgccttgcg tataatattt gcccatggtg aaaacggggg cgaagaagtt gtccatattg
     4561 gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata
     4621 ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc
     4681 gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac
     4741 gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc
     4801 tcaccgtctt tcattgccat acgaaattcc ggatgagcat tcatcaggcg ggcaagaatg
     4861 tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta
     4921 atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa
     4981 tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc
     5041 attttagctt ccttagctcc tgaaaatctc gccaagctag cttggattct caccaataaa
     5101 aaacgcccgg cggcaaccga gcgttctgaa caaatccaga tggagttctg aggtcattac
     5161 tggatctatc aacaggagtc caagctcagc taattaagct tggctgcagg tcgacccggg
     5221 gtaccttaag atctacctcc tgaacctccc tccgaccact gattgacaat gcgctgcatc
     5281 cccttcacgt ggtagaggat gtcgtccagc tctgggaaga tgtcctcgtt ggatctagaa
     5341 cctcctgaac ctccggatct cttgtacagc tcgtccatgc cgagagtgat cccggcggcg
     5401 gtcacgaact ccagcaggac catgtgatcg cgcttctcgt tggggtcttt gctcagcttg
     5461 gactgggtgc tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg
     5521 ttctgctggt agtggtcggc gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg
     5581 aagttcac
//
