LOCUS       sequence-343747-        6247 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HaloTag7 at the at the Golgi apparatus of mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6247)
  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 6247)
  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_Beta4Gal-HaloTag7
FEATURES             Location/Qualifiers
     source          1..6247
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(246..1037,1038..1106))
                     /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     869..888
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1107..1211)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1286..1305)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1477..1856
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1857..2060
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2011..2031
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2062..2080
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2083..2101
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2111..2135
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2513..3397
                     /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(3441..3458)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3447..3671
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3804..3823)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3955..4002
                     /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             4010..5032
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4010..5032
                     /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    5162..5283
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5199..5218)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5253..5272
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5332..5348)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5332..5348)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5345..5367)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5356..5372
                     /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(5380..5386,5387..5404,5405..5410))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5425..5446
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5563..5580)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5734..6247,1..75))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5814..5833)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
       61 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
      121 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
      181 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
      241 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
      301 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
      361 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
      421 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
      481 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
      541 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
      601 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
      661 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
      721 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
      781 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
      841 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
      901 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
      961 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     1021 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     1081 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     1141 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     1201 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
     1261 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     1321 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
     1381 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
     1441 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
     1501 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     1561 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     1621 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     1681 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     1741 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     1801 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     1861 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     1921 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     1981 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     2041 aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc agtgatagag
     2101 atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt
     2161 ttgacctcca tagaagacac cgggaccgat ccagcctccg gactctagcg tttaaactta
     2221 agcttggtac cgagctcgga tccactagtc cagtgtggtg gaattctgca gatatccagc
     2281 aaccatgagg cttcgggagc cgctcctgag cggcagcgcc gcgatgccag gcgcgtccct
     2341 acagcgggcc tgccgcctgc tcgtggccgt ctgcgctctg caccttggcg tcaccctcgt
     2401 ttactacctg gctggccgcg acctgagccg cctgccccaa ctggtcggag tctccacacc
     2461 gctgcagtcg acggtaccgc gggcccggga tccaccggtc gccaccggat ccgaaatcgg
     2521 tactggcttt ccattcgacc cccattatgt ggaagtcctg ggcgagcgca tgcactacgt
     2581 cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc cgacctcctc
     2641 ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc catcgctgca ttgctccaga
     2701 cctgatcggt atgggcaaat ccgacaaacc agacctgggt tatttcttcg acgaccacgt
     2761 ccgcttcatg gatgccttca tcgaagccct gggtctggaa gaggtcgtcc tggtcattca
     2821 cgactggggc tccgctctgg gtttccactg ggccaagcgc aatccagagc gcgtcaaagg
     2881 tattgcattt atggagttca tccgccctat cccgacctgg gacgaatggc cagaatttgc
     2941 ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc cgcaagctga tcatcgatca
     3001 gaacgttttt atcgagggta cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga
     3061 gatggaccat taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt
     3121 cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata
     3181 catggactgg ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt
     3241 tctgatccca ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt
     3301 ggacatcggc ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga
     3361 gatcgcgcgc tggctgtcta ctctggagat ttccggttaa ccgctcgagt ctagagggcc
     3421 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3481 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3541 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3601 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3661 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     3721 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3781 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3841 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3901 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta cctagaagtt
     3961 cctattccga agttcctatt ctctagaaag tataggaact tccttggcca aaaagcctga
     4021 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct
     4081 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg
     4141 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg
     4201 gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga
     4261 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga
     4321 aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc
     4381 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac
     4441 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt
     4501 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc
     4561 cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct
     4621 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc
     4681 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca
     4741 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta
     4801 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga
     4861 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg
     4921 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact
     4981 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agcacgtact
     5041 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     5101 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     5161 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     5221 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5281 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     5341 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5401 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5461 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5521 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5581 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5641 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5701 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5761 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5821 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5881 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5941 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6001 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6061 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6121 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6181 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6241 atccggc
//
