LOCUS       sequence-190571-        6218 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses C-Terminal segment of C. elegans Piezo1 with M2225R 
            mutation in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6218)
  AUTHORS   Kamajaya A, Kaiser JT, Lee J, Reid M, Rees DC
  TITLE     The structure of a conserved piezo channel domain reveals a 
            topologically distinct beta sandwich fold.
  JOURNAL   Structure. 2014 Oct 7;22(10):1520-7. doi: 10.1016/j.str.2014.08.009.
            Epub 2014 Sep 18.
  PUBMED    25242456
REFERENCE   2  (bases 1 to 6218)
  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: 
            "10.1016/j.str.2014.08.009"; journalName: "Structure"; date: 
            "2014-10-7- 7"; volume: "22"; issue: "10"; pages: "1520-7"
COMMENT     Sequence Label: CTL2 (M2225R)
FEATURES             Location/Qualifiers
     source          1..6218
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      6..461
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(93..112)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     303..324
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        487..591
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(592..660,661..1452)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPXAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(810..829)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1623..2211
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2112..2131
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2365..2382
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2397..2536
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2462..2481)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2622..2644
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2638..2829)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3638..4720)
                     /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     4682..4701
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4721..4798)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5004..5023
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5107..5126
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5107..5125
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5126..5150
                     /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             5165..5187
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6056..6073
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6126..6144)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6140..6187
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
       61 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
      121 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
      181 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
      241 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
      301 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
      361 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
      421 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
      481 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
      541 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
      601 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
      661 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
      721 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
      781 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
      841 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
      901 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
      961 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     1021 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     1081 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgyagca
     1141 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     1201 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     1261 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     1321 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     1381 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     1441 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     1501 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     1561 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     1621 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     1681 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     1741 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     1801 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     1861 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     1921 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     1981 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     2041 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     2101 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     2161 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     2221 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     2281 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     2341 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     2401 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     2461 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     2521 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     2581 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     2641 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     2701 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     2761 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     2821 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     2881 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     2941 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     3001 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     3061 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     3121 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     3181 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     3241 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     3301 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     3361 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     3421 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     3481 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     3541 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     3601 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3661 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3721 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     3781 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     3841 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     3901 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     3961 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     4021 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     4081 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     4141 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     4201 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     4261 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     4321 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     4381 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     4441 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     4501 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     4561 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     4621 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     4681 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     4741 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     4801 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     4861 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     4921 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     4981 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     5041 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     5101 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
     5161 taattttgtt taactttaag aaggagatat acaaggaggt atgtcgctac tcaatcaaat
     5221 cggaactatc agtatgccag aaaaagttac gttgaggatt tcgattgaag gatatccacc
     5281 actttacgag agggaggcac aaggaagtaa tcacgataac gctgaacttg gaatgataaa
     5341 acctgatcaa ttggcatctt tgaatcaagc attgacggat agttacacga cacgagacac
     5401 taattctatt ttaagatcaa gaatgtctgt atcatatctg aaaggataca catatgagga
     5461 tattctgatt gtgcgattcc gtccagaatc tgaaatttat tggccgattt cacaggattc
     5521 gagaaatgcg atgatcgata aattgagccg aaacacgtca gtcaattttg aagtatctct
     5581 ggaattcact cggccatatg atccaaatga aaacgctgct ttaaaacatt cgaaatcgtg
     5641 gcttgttcca atctccctgg atatgacgat tcgagcaaaa attcaaagtg ctctccgagg
     5701 agatccaggt catccgattc tcattccaca gtcaattcct gcgtttattc aagttccaaa
     5761 tcaaggagaa ttgacccttc caacatcaat cggaaatact attatcaatg acggaaatcc
     5821 acggattaat acgaccggga tggaaaaatc agacgaagct cgggcttggt tcgactcgtt
     5881 aaccttgaat ctcgaacaag gaaaatcgca aaacgagaaa atgtggatcg ccacatccga
     5941 acatccagga gaccaaaatg cgaaactctg gataaagact gcaaatacga cgtattcggg
     6001 aaggccgtac cttcaagttg tcggatttat tgatagagca ttcccatcac tcgagcacca
     6061 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     6121 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     6181 ttttttgctg aaaggaggaa ctatatccgg attggcga
//
