LOCUS       sequence-390373-        6807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of cytosolic metastable Halotag/folding sensor 
            (Cyto HaloK73T).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6807)
  AUTHORS   Melo EP, Konno T, Farace I, Awadelkareem MA, Skov LR, Teodoro F, 
            Sancho TP, Paton AW, Paton JC, Fares M, Paulo PMR, Zhang X, Avezov E
  TITLE     Stress-induced protein disaggregation in the endoplasmic reticulum 
            catalysed by BiP.
  JOURNAL   Nat Commun. 2022 May 6;13(1):2501. doi: 10.1038/s41467-022-30238-2.
  PUBMED    35523806
REFERENCE   2  (bases 1 to 6807)
  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: "Nat 
            Commun."; date: "2022-05-6"; volume: "13(1):2501. doi"; pages: " 
            10.1038/s41467-022-30238-2"
COMMENT     Sequence Label: pCMV_HaloK73T_cyto
FEATURES             Location/Qualifiers
     source          1..6807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             618..635
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    2831..3453
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3030..3049)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(3477..3497)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3482..3811
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3662..3797
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3849..3868)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3850..3868)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3882..3898)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3882..3898)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3895..3917)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3906..3922
                     /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(3930..3936,3937..3954,3955..3960))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3975..3996
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4113..4130)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4284..4872)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4364..4383)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5043..5834,5835..5903))
                     /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     5666..5685
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5904..6008)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6035..6490
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6122..6141)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6332..6353
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6616..6638
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6630..6647
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6631..6647
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agaattaatt caccatgcac catcatcatc accatgcggc cgcatctctc gaggcagaaa
      661 tcggtactgg ctttccattc gacccccatt atgtggaagt cctgggcgag cgcatgcact
      721 acgtcgatgt tggtccgcgc gatggcaccc ctgtgctgtt cctgcacggt aacccgacct
      781 cctcctacgt gtggcgcaac atcatcccgc atgttgcacc gacccatcgc tgcattgctc
      841 cagacctgat cggtatgggc aaatccgaca caccagacct gggttatttc ttcgacgacc
      901 acgtccgctt catggatgcc ttcatcgaag ccctgggtct ggaagaggtc gtcctggtca
      961 ttcacgactg gggctccgct ctgggtttcc actgggccaa gcgcaatcca gagcgcgtca
     1021 aaggtattgc atttatggag ttcatccgcc ctatcccgac ctgggacgaa tggccagaat
     1081 ttgcccgcga gaccttccag gccttccgca ccaccgacgt cggccgcaag ctgatcatcg
     1141 atcagaacgt ttttatcgag ggtacgctgc cgatgggtgt cgtccgcccg ctgactgaag
     1201 tcgagatgga ccattaccgc gagccgttcc tgaatcctgt tgaccgcgag ccactgtggc
     1261 gcttcccaaa cgagctgcca atcgccggtg agccagcgaa catcgtcgcg ctggtcgaag
     1321 aatacatgga ctggctgcac cagtcccctg tcccgaagct gctgttctgg ggcaccccag
     1381 gcgttctgat cccaccggcc gaagccgctc gcctggccaa aagcctgcct aactgcaagg
     1441 ctgtggacat cggcccgggt ctgaatctgc tgcaagaaga caacccggac ctgatcggca
     1501 gcgagatcgc gcgctggctt tcgacggtcg acggatccgc agatctagcg gccaaggatg
     1561 agttgtagtc cgtgccgctg ctgctcggcc tcctcggcct ggccgtcgcc gagcctgccg
     1621 tctacttcaa ggagcagttt ctggacggag acgggtggac ttcccgctgg atcgaatcca
     1681 aacacaagtc agattttggc aaattcgttc tcagttccgg caagttctac ggtgacgagg
     1741 agaaagataa aggtttgcag acaagccagg atgcacgctt ttatgctctg tcggccagtt
     1801 tcgagccttt cagcaacaaa ggccagacgc tggtggtgca gttcacggtg aaacatgagc
     1861 agaacatcga ctgtgggggc ggctatgtga agctgtttcc taatagtttg gaccagacag
     1921 acatgcacgg agactcagaa tacaacatca tgtttggtcc cgacatctgt ggccctggca
     1981 ccaagaaggt tcatgtcatc ttcaactaca agggcaagaa cgtgctgatc aacaaggaca
     2041 tccgttgcaa ggatgatgag tttacacacc tgtacacact gattgtgcgg ccagacaaca
     2101 cctatgaggt gaagattgac aacagccagg tggagtccgg ctccttggaa gacgattggg
     2161 acttcctgcc acccaagaag ataaaggatc ctgatgcttc aaaaccggaa gactgggatg
     2221 agcgggccaa gatcgatgat cccacagact ccaagcctga ggactgggac aagcccgagc
     2281 atatccctga ccctgatgct aagaagcccg aggactggga tgaagagatg gacggagagt
     2341 gggaaccccc agtgattcag aaccctgagt acaagggtga gtggaagccc cggcagatcg
     2401 acaacccaga ttacaagggc acttggatcc acccagaaat tgacaacccc gagtattctc
     2461 ccgatcccag tatctatgcc tatgataact ttggcgtgct gggcctggac ctctggcagg
     2521 tcaagtctgg caccatcttt gacaacttcc tcatcaccaa cgatgaggca tacgctgagg
     2581 agtttggcaa cgagacgtgg ggcgtaacaa aggcagcaga gaaacaaatg aaggacaaac
     2641 aggacgagga gcagaggaca aggaggatga tgaggacaaa gatgaggatg aggaggatga
     2701 ggaggacaag gaggaagatg aggaggaaga tgtccccggc caggccaagg acgagctgta
     2761 gagaggcctg cctccagggc tggactgagg cctgaactag tcgcggccgc tttcgaatct
     2821 agaggatccc gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt
     2881 tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga
     2941 ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt
     3001 tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc
     3061 acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc
     3121 tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg
     3181 gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat
     3241 ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc
     3301 tgtccttctg attttaaaat aactatacca gcaggaggac gtccagacac agcataggct
     3361 acctggccat gcccaaccgg tgggacattt gagttgcttg cttggcactg tcctctcatg
     3421 cgttgggtcc actcagtaga tgcctgttga attgggtacg cggccagctt ggctgtggaa
     3481 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3541 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     3601 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     3661 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3721 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     3781 aggctttttt ggaggcctag gcttttgcaa aaagctcctc gaggaactga aaaaccagaa
     3841 agttaattcc ctatagtgag tcgtattaaa ttcgtaatca tgtcatagct gtttcctgtg
     3901 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     3961 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4021 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4081 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4141 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4201 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4261 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4321 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4381 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4441 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4501 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4561 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4621 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4681 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4741 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4801 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4861 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4921 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4981 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5041 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5101 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5161 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5221 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5281 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5341 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5401 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5461 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5521 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5581 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5641 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5701 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5761 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5821 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5881 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5941 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6001 gttccgcgca catttccccg aaaagtgcca cctgacgcgc cctgtagcgg cgcattaagc
     6061 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     6121 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     6181 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     6241 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     6301 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     6361 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     6421 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     6481 cttacaattt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     6541 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     6601 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct
     6661 atacattgaa tcaatattgg caattagcca tattagtcat tggttatata gcataaatca
     6721 atattggcta ttggccattg catacgttgt atctatatca taatatgtac atttatattg
     6781 gctcatgtcc aatatgaccg ccatgtt
//
