LOCUS       sequence-390336-        6852 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of ER-targeted metastable Halotag/folding 
            sensor (HaloK73T).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6852)
  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 6852)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: SP_H6_HaloK73T_KDEL_pCMV1
FEATURES             Location/Qualifiers
     source          1..6852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     122..144
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     136..153
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     137..153
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        314..693
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        694..897
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     848..868
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             928..972
                     /codon_start=1
                     /product="leader sequence from mouse preprotrypsin"
                     /label=preprotrypsin leader
                     /translation="MSALLILALVGAAVA"
     CDS             976..993
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    3189..3811
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3388..3407)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(3835..3855)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3840..4169
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4020..4155
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4207..4226)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4208..4226)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4240..4256)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4240..4256)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4253..4275)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4264..4280
                     /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(4288..4294,4295..4312,4313..4318))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4333..4354
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4471..4488)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4642..5230)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4722..4741)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5401..6192,6193..6261))
                     /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     6024..6043
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6262..6366)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6393..6848
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6480..6499)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6690..6711
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
       61 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
      121 tcccagtcac gacgttgtaa aacgacggcc agtgccaagc tgatctatac attgaatcaa
      181 tattggcaat tagccatatt agtcattggt tatatagcat aaatcaatat tggctattgg
      241 ccattgcata cgttgtatct atatcataat atgtacattt atattggctc atgtccaata
      301 tgaccgccat gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      361 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      421 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      481 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      541 ttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt caatgacggt
      601 aaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc tacttggcag
      661 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacaccaat
      721 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      781 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa taaccccgcc
      841 ccgttgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      901 ttagtgaacc gtcagaatta attcaccatg tctgcacttc tgatcctagc tcttgttgga
      961 gctgcagttg ctatgcacca tcatcatcac catgcggccg catctctcga ggcagaaatc
     1021 ggtactggct ttccattcga cccccattat gtggaagtcc tgggcgagcg catgcactac
     1081 gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa cccgacctcc
     1141 tcctacgtgt ggcgcaacat catcccgcat gttgcaccga cccatcgctg cattgctcca
     1201 gacctgatcg gtatgggcaa atccgacaca ccagacctgg gttatttctt cgacgaccac
     1261 gtccgcttca tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt cctggtcatt
     1321 cacgactggg gctccgctct gggtttccac tgggccaagc gcaatccaga gcgcgtcaaa
     1381 ggtattgcat ttatggagtt catccgccct atcccgacct gggacgaatg gccagaattt
     1441 gcccgcgaga ccttccaggc cttccgcacc accgacgtcg gccgcaagct gatcatcgat
     1501 cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg tccgcccgct gactgaagtc
     1561 gagatggacc attaccgcga gccgttcctg aatcctgttg accgcgagcc actgtggcgc
     1621 ttcccaaacg agctgccaat cgccggtgag ccagcgaaca tcgtcgcgct ggtcgaagaa
     1681 tacatggact ggctgcacca gtcccctgtc ccgaagctgc tgttctgggg caccccaggc
     1741 gttctgatcc caccggccga agccgctcgc ctggccaaaa gcctgcctaa ctgcaaggct
     1801 gtggacatcg gcccgggtct gaatctgctg caagaagaca acccggacct gatcggcagc
     1861 gagatcgcgc gctggctttc gacggtcgac ggatccgcag atctagcggc caaggatgag
     1921 ttgtagtccg tgccgctgct gctcggcctc ctcggcctgg ccgtcgccga gcctgccgtc
     1981 tacttcaagg agcagtttct ggacggagac gggtggactt cccgctggat cgaatccaaa
     2041 cacaagtcag attttggcaa attcgttctc agttccggca agttctacgg tgacgaggag
     2101 aaagataaag gtttgcagac aagccaggat gcacgctttt atgctctgtc ggccagtttc
     2161 gagcctttca gcaacaaagg ccagacgctg gtggtgcagt tcacggtgaa acatgagcag
     2221 aacatcgact gtgggggcgg ctatgtgaag ctgtttccta atagtttgga ccagacagac
     2281 atgcacggag actcagaata caacatcatg tttggtcccg acatctgtgg ccctggcacc
     2341 aagaaggttc atgtcatctt caactacaag ggcaagaacg tgctgatcaa caaggacatc
     2401 cgttgcaagg atgatgagtt tacacacctg tacacactga ttgtgcggcc agacaacacc
     2461 tatgaggtga agattgacaa cagccaggtg gagtccggct ccttggaaga cgattgggac
     2521 ttcctgccac ccaagaagat aaaggatcct gatgcttcaa aaccggaaga ctgggatgag
     2581 cgggccaaga tcgatgatcc cacagactcc aagcctgagg actgggacaa gcccgagcat
     2641 atccctgacc ctgatgctaa gaagcccgag gactgggatg aagagatgga cggagagtgg
     2701 gaacccccag tgattcagaa ccctgagtac aagggtgagt ggaagccccg gcagatcgac
     2761 aacccagatt acaagggcac ttggatccac ccagaaattg acaaccccga gtattctccc
     2821 gatcccagta tctatgccta tgataacttt ggcgtgctgg gcctggacct ctggcaggtc
     2881 aagtctggca ccatctttga caacttcctc atcaccaacg atgaggcata cgctgaggag
     2941 tttggcaacg agacgtgggg cgtaacaaag gcagcagaga aacaaatgaa ggacaaacag
     3001 gacgaggagc agaggacaag gaggatgatg aggacaaaga tgaggatgag gaggatgagg
     3061 aggacaagga ggaagatgag gaggaagatg tccccggcca ggccaaggac gagctgtaga
     3121 gaggcctgcc tccagggctg gactgaggcc tgaactagtc gcggccgctt tcgaatctag
     3181 aggatcccgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg
     3241 ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact
     3301 aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg
     3361 ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac
     3421 aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc
     3481 ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt
     3541 agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct
     3601 acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg
     3661 tccttctgat tttaaaataa ctataccagc aggaggacgt ccagacacag cataggctac
     3721 ctggccatgc ccaaccggtg ggacatttga gttgcttgct tggcactgtc ctctcatgcg
     3781 ttgggtccac tcagtagatg cctgttgaat tgggtacgcg gccagcttgg ctgtggaatg
     3841 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3901 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3961 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     4021 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     4081 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     4141 gcttttttgg aggcctaggc ttttgcaaaa agctcctcga ggaactgaaa aaccagaaag
     4201 ttaattccct atagtgagtc gtattaaatt cgtaatcatg tcatagctgt ttcctgtgtg
     4261 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4321 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4381 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4441 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4501 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4561 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4621 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4681 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4741 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4801 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4861 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4921 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4981 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5041 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5101 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5161 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5221 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5281 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5341 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5401 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5461 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5521 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5581 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5641 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5701 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5761 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5821 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5881 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5941 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6001 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6061 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6121 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6181 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6241 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6301 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6361 tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc
     6421 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     6481 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     6541 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     6601 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     6661 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     6721 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     6781 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     6841 tacaatttcc at
//
