LOCUS       sequence-343740-        6247 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HaloTag9 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_b4g-HaloTag9
FEATURES             Location/Qualifiers
     source          1..6247
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(25..44)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        216..595
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        596..799
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     750..770
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    801..819
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    822..840
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     850..874
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1252..2136
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDH
                     NVFIEGTLRMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     primer_bind     complement(2180..2197)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2186..2410
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2543..2562)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2694..2741
                     /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             2749..3771
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2749..3771
                     /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    3901..4022
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3938..3957)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3992..4011
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4071..4087)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4071..4087)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4084..4106)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4095..4111
                     /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(4119..4125,4126..4143,4144..4149))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4164..4185
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4302..4319)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4473..5061)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4553..4572)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5232..6023,6024..6092))
                     /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     5855..5874
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6093..6197)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
       61 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
      121 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
      181 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
      241 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      301 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      361 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      421 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      481 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      541 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
      601 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
      661 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
      721 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
      781 ggtctatata agcagagctc tccctatcag tgatagagat ctccctatca gtgatagaga
      841 tcgtcgacga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt
      901 tgacctccat agaagacacc gggaccgatc cagcctccgg actctagcgt ttaaacttaa
      961 gcttggtacc gagctcggat ccactagtcc agtgtggtgg aattctgcag atatccagca
     1021 accatgaggc ttcgggagcc gctcctgagc ggcagcgccg cgatgccagg cgcgtcccta
     1081 cagcgggcct gccgcctgct cgtggccgtc tgcgctctgc accttggcgt caccctcgtt
     1141 tactacctgg ctggccgcga cctgagccgc ctgccccaac tggtcggagt ctccacaccg
     1201 ctgcagtcga cggtaccgcg ggcccgggat ccaccggtcg ccaccggatc cgaaatcggt
     1261 actggctttc cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat gcactacgtc
     1321 gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc gacctcctcc
     1381 tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat tgctccagac
     1441 ctgatcggta tgggcaaatc cgacaaacca gacctgggtt atttcttcga cgaccacgtc
     1501 cgcttcatgg atgccttcat cgaagccctg ggtctggaag aggtcgtcct ggtcattcac
     1561 gactggggct ccgctctggg tttccactgg gccaagcgca atccagagcg cgtcaaaggt
     1621 attgcattta tggagttcat ccgccctatc ccgacctggg acgaatggcc agaatttgcc
     1681 cgcgagacct tccaggcctt ccgcaccacc gacgtcggcc gcaagctgat catcgatcat
     1741 aacgttttta tcgagggtac gctgcgtatg ggtgtcgtcc gcccgctgac tgaagtcgag
     1801 atggaccatt accgcgagcc gttcctgaat cctgttgacc gcgagccact gtggcgcttc
     1861 ccaaacgagc tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt cgaagaatac
     1921 atggactggc tgcaccagtc ccctgtcccg aagctgctgt tctggggcac cccaggcgtt
     1981 ctgatcccac cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg caaggctgtg
     2041 gacatcggcc cgggtctgaa tctgctgcaa gaagacaacc cggacctgat cggcagcgag
     2101 atcgcgcgct ggctgtctac tctggagatt tccggttaac cgctcgagtc tagagggccc
     2161 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2221 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2281 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2341 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     2401 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     2461 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2521 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2581 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2641 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac ctagaagttc
     2701 ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa aaagcctgaa
     2761 ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg
     2821 atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga
     2881 tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg
     2941 cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag
     3001 agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa
     3061 accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc
     3121 gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact
     3181 acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg
     3241 atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc
     3301 gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg
     3361 acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc
     3421 caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag
     3481 acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat
     3541 atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat
     3601 gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg
     3661 cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc
     3721 gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata gcacgtacta
     3781 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     3841 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     3901 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     3961 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4021 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     4081 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4141 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4201 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4261 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     4321 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4381 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4441 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4501 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4561 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4621 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4681 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4741 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4801 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4861 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4921 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4981 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5041 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5101 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5161 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5221 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5281 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5341 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5401 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5461 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5521 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5581 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5641 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5701 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5761 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5821 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5881 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5941 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6001 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6061 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6121 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6181 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     6241 gatctcc
//
