LOCUS       sequence-307683-        6134 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Express C49S/C117S double mutant of Zebrafish Halo-Tev-HSPB7 in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6134)
  AUTHORS   Surya SL, Long MJC, Urul DA, Zhao Y, Mercer EJ, EIsaid IM, Evans T, 
            Aye Y
  TITLE     Cardiovascular Small Heat Shock Protein HSPB7 Is a Kinetically 
            Privileged Reactive Electrophilic Species (RES) Sensor.
  JOURNAL   ACS Chem Biol. 2018 Feb 8. doi: 10.1021/acschembio.7b00925.
  PUBMED    29397684
REFERENCE   2  (bases 1 to 6134)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Chem 
            Biol. 2018 Feb 8. doi: 10.1021/acschembio.7b00925."
COMMENT     Sequence Label: pCS2 Halo-Tev- HSPB7 (zebrafish) C49S and C117S
FEATURES             Location/Qualifiers
     source          1..6134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             267..1157
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             1158..1178
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     polyA_signal    2186..2320
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2210..2229)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2264..2283
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2433..2453)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2433..2451)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2472..2488)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2472..2488)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2485..2507)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2496..2512
                     /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(2520..2526,2527..2544,2545..2550))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2565..2586
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2703..2720)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2874..3462)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2954..2973)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3633..4424,4425..4493))
                     /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     4256..4275
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4494..4598)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4624..5079)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4761..4782)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4973..4992
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5150..6134
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgaattca aggcctctcg agcactgttg
      121 gcctactgga caagcaggca gacccagctc atataaacac tgaaaaagct gctgagtgga
      181 ttcatggtgt tttgaataag aacttgatca ccggttcaat ttgaagttct gactggatat
      241 cgatttgaga aagcatctca acactaatgg cagaaatcgg tactggcttt ccattcgacc
      301 cccattatgt ggaagtcctg ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg
      361 gcacccctgt gctgttcctg cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca
      421 tcccgcatgt tgcaccgacc catcgctgca ttgctccaga cctgatcggt atgggcaaat
      481 ccgacaaacc agacctgggt tatttcttcg acgaccacgt ccgcttcatg gatgccttca
      541 tcgaagccct gggtctggaa gaggtcgtcc tggtcattca cgactggggc tccgctctgg
      601 gtttccactg ggccaagcgc aatccagagc gcgtcaaagg tattgcattt atggagttca
      661 tccgccctat cccgacctgg gacgaatggc cagaatttgc ccgcgagacc ttccaggcct
      721 tccgcaccac cgacgtcggc cgcaagctga tcatcgatca gaacgttttt atcgagggta
      781 cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat taccgcgagc
      841 cgttcctgaa ccctgttgac cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg
      901 ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata catggactgg ctgcaccagt
      961 cccctgtccc gaagctgctg ttctggggca ccccaggcgt tctgatccca ccggccgaag
     1021 ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc ccgggtctga
     1081 atctgctgca agaagacaac ccggacctga tcggcagcga gatcgcgcgc tggctgtcga
     1141 cgctcgagat ttccggcgaa aacttgtatt tccagggctc aatgagcgcg agcaattctt
     1201 ctgcctatcg atcggagcgt agttaccatc agacgtcatc ctcctcatct tcatcctcta
     1261 cctctgcaaa cccatacatg gagaagagcc gaggactctt cgctgacgac tttggatctt
     1321 tcatgagtcc gaaggatgct ttgggatttc caaacagaac aggcactgtt ggaaacatca
     1381 agactcttgg agacacatac cagttcacag tggacgtgca agacttctcg cctgaggatg
     1441 tgatcgtcac cacctcaaac aatcagattg aagttcatgc tgaaaagctt gctagcgatg
     1501 ggacagtgat gaacactttc acccataaaa gccggctacc agaagatgtg gaccccactt
     1561 cagtgaagtc cagtttaggg gccgatggga ctctgactat aaaagcacag agaaacacag
     1621 ctaaactgga gcatgcacag acgttccgca cagagatcaa aatctagacc tgtgccctcc
     1681 tgctgtgtct gtttcactgt gataaatgac cttgaaaata tagctactta gccctgcttt
     1741 gtcattcctc catgaccttc tctcatttct gctcattaga cttggatgga atttaatcct
     1801 gtcacatgta ttcaaaacct ttaagatgaa tgcactgcta acgtcagctc tctgctaatg
     1861 acatatcagc ttacaggaca aagtgggctc caaaattctg gaagtacatt taatccaaaa
     1921 tataattgaa acattgaaag atatcagaat atatatggct atacagtaga tatagtactt
     1981 tagtacttta aattacattt agttcaccca aacatgtaaa tcctgccatt atttactcat
     2041 ttccacttgt tccaaaccca attaagttcc gttgaacaca aaataaaatg tgcaatgatg
     2101 gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaarrc cacatgtgct cgagcctcta
     2161 gaactatagt gagtcgtatt acgtagatcc agacatgata agatacattg atgagtttgg
     2221 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     2281 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     2341 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taattcgcgg ccgcggcgcc
     2401 aatgcattgg gcccggtacc cagcttttgt tccctttagt gagggttaat tgcgcgcttg
     2461 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     2521 aacatacgag ccgggagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     2581 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     2641 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     2701 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     2761 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2821 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2881 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2941 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3001 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3061 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     3121 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     3181 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3241 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     3301 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3361 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3421 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3481 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3541 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3601 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3661 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3721 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     3781 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     3841 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3901 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3961 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     4021 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     4081 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     4141 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     4201 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     4261 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     4321 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4381 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4441 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4501 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     4561 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     4621 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     4681 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     4741 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     4801 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     4861 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     4921 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     4981 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     5041 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc
     5101 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag tcgaccgcca
     5161 attcaatatg gcgtatatgg actcatgcca attcaatatg gtggatctgg acctgtgcca
     5221 attcaatatg gcgtatatgg actcgtgcca attcaatatg gtggatctgg accccagcca
     5281 attcaatatg gcggacttgg caccatgcca attcaatatg gcggacctgg cactgtgcca
     5341 actggggagg ggtctacttg gcacggtgcc aagtttgagg aggggtcttg gccctgtgcc
     5401 aagtccgcca tattgaattg gcatggtgcc aataatggcg gccatattgg ctatatgcca
     5461 ggatcaatat ataggcaata tccaatatgg ccctatgcca atatggctat tggccaggtt
     5521 caatactatg tattggccct atgccatata gtattccata tatgggtttt cctattgacg
     5581 tagatagccc ctcccaatgg gcggtcccat ataccatata tggggcttcc taataccgcc
     5641 catagccact cccccattga cgtcaatggt ctctatatat ggtctttcct attgacgtca
     5701 tatgggcggt cctattgacg tatatggcgc ctcccccatt gacgtcaatt acggtaaatg
     5761 gcccgcctgg ctcaatgccc attgacgtca ataggaccac ccaccattga cgtcaatggg
     5821 atggctcatt gcccattcat atccgttctc acgcccccta ttgacgtcaa tgacggtaaa
     5881 tggcccactt ggcagtacat caatatctat taatagtaac ttggcaagta cattactatt
     5941 ggaagtacgc cagggtacat tggcagtact cccattgacg tcaatggcgg taaatggccc
     6001 gcgatggctg ccaagtacat ccccattgac gtcaatgggg aggggcaatg acgcaaatgg
     6061 gcgttccatt gacgtaaatg ggcggtaggc gtgcctaatg ggaggtctat ataagcaatg
     6121 ctcgtttagg gaac
//
