LOCUS       sequence-343754-        6151 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HaloTag7 in the endoplasmic reticulum of mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6151)
  AUTHORS   Frei MS, Tarnawski M, Roberti MJ, Koch B, Hiblot J, Johnsson K
  TITLE     Engineered HaloTag variants for fluorescence lifetime multiplexing.
  JOURNAL   Nat Methods. 2021 Dec 16. pii: 10.1038/s41592-021-01341-x. doi: 
            10.1038/s41592-021-01341-x.
  PUBMED    34916672
REFERENCE   2  (bases 1 to 6151)
  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 
            Methods. 2021 Dec 16. pii: 10.1038/s41592-021-01341-x. doi: 
            10.1038/s41592-021-01341-x."
COMMENT     Sequence Label: pcDNA5/FRT/TO_CalR_HaloTag7_KDEL
FEATURES             Location/Qualifiers
     source          1..6151
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        70..449
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        450..653
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     604..624
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    655..673
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    676..694
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     704..728
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             965..1849
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     primer_bind     complement(1938..1955)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1944..2168
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2301..2320)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2452..2499
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2507..3529
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2507..3529
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3659..3780
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3696..3715)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3750..3769
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3829..3845)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3829..3845)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3842..3864)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3853..3869
                     /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(3877..3883,3884..3901,3902..3907))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3922..3943
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4060..4077)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4231..4819)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4311..4330)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4990..5781,5782..5850))
                     /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     5613..5632
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5851..5955)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6030..6049)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
       61 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
      121 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      181 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      241 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      301 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      361 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      421 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      481 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      541 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      601 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctcccta
      661 tcagtgatag agatctccct atcagtgata gagatcgtcg acgagctcgt ttagtgaacc
      721 gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc
      781 gatccagcct ccggactcta gcgtttaaac ttaagcttgg taccgagctc ggatccacta
      841 gtccagtgtg gtggaattct gcagatatcc agcaaccatg ctgctatccg tgccgttgct
      901 gctcggcctc ctcggcctgg ccgtcgccgg aggcagcgga ggcagcgaat tcggatccgg
      961 atccgaaatc ggtactggct ttccattcga cccccattat gtggaagtcc tgggcgagcg
     1021 catgcactac gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa
     1081 cccgacctcc tcctacgtgt ggcgcaacat catcccgcat gttgcaccga cccatcgctg
     1141 cattgctcca gacctgatcg gtatgggcaa atccgacaaa ccagacctgg gttatttctt
     1201 cgacgaccac gtccgcttca tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt
     1261 cctggtcatt cacgactggg gctccgctct gggtttccac tgggccaagc gcaatccaga
     1321 gcgcgtcaaa ggtattgcat ttatggagtt catccgccct atcccgacct gggacgaatg
     1381 gccagaattt gcccgcgaga ccttccaggc cttccgcacc accgacgtcg gccgcaagct
     1441 gatcatcgat cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg tccgcccgct
     1501 gactgaagtc gagatggacc attaccgcga gccgttcctg aatcctgttg accgcgagcc
     1561 actgtggcgc ttcccaaacg agctgccaat cgccggtgag ccagcgaaca tcgtcgcgct
     1621 ggtcgaagaa tacatggact ggctgcacca gtcccctgtc ccgaagctgc tgttctgggg
     1681 caccccaggc gttctgatcc caccggccga agccgctcgc ctggccaaaa gcctgcctaa
     1741 ctgcaaggct gtggacatcg gcccgggtct gaatctgctg caagaagaca acccggacct
     1801 gatcggcagc gagatcgcgc gctggctgtc tactctggag atttccggtg gcgctccagg
     1861 attctcctca atttccgcta aagatgaact gtagtaaccg ctcgagtcta gagggcccgt
     1921 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     1981 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2041 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2101 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     2161 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     2221 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2281 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2341 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2401 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct
     2461 attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact
     2521 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat
     2581 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata
     2641 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca
     2701 ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag
     2761 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac
     2821 cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga
     2881 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac
     2941 atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat
     3001 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga
     3061 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac
     3121 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca
     3181 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac
     3241 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat
     3301 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc
     3361 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg
     3421 tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc
     3481 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg
     3541 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     3601 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     3661 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     3721 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     3781 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     3841 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     3901 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     3961 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4021 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4081 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4141 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4201 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4261 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4321 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4381 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4441 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4501 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4561 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4621 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4681 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4741 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4801 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4861 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4921 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4981 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5041 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5101 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5161 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5221 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5281 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     5341 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     5401 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     5461 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     5521 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     5581 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     5641 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5701 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     5761 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     5821 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     5881 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5941 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     6001 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     6061 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
     6121 gctacaacaa ggcaaggctt gaccgacaat t
//
