LOCUS       sequence-343742-        6070 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Peroxysomal expression of HaloTag9 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6070)
  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 6070)
  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_HaloTag9_SKL
FEATURES             Location/Qualifiers
     source          1..6070
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(62..81)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        253..632
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        633..836
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     787..807
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    838..856
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    859..877
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     887..911
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1070..1954
                     /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(2040..2057)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2046..2270
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2403..2422)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2554..2601
                     /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             2609..3631
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2609..3631
                     /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    3761..3882
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3798..3817)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3852..3871
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3931..3947)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3931..3947)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3944..3966)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3955..3971
                     /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(3979..3985,3986..4003,4004..4009))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4024..4045
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4162..4179)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4333..4921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4413..4432)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5092..5883,5884..5952))
                     /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     5715..5734
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5953..6057)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
       61 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
      121 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
      181 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
      241 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      301 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      361 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      421 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      481 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      541 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      601 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      661 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      721 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      781 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctcc
      841 ctatcagtga tagagatctc cctatcagtg atagagatcg tcgacgagct cgtttagtga
      901 accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg
      961 accgatccag cctccggact ctagcgttta aacttaagct tggtaccgag ctcggatcca
     1021 ctagtccagt gtggtggaat tctgcagata tccagcaacc atgggatccg aaatcggtac
     1081 tggctttcca ttcgaccccc attatgtgga agtcctgggc gagcgcatgc actacgtcga
     1141 tgttggtccg cgcgatggca cccctgtgct gttcctgcac ggtaacccga cctcctccta
     1201 cgtgtggcgc aacatcatcc cgcatgttgc accgacccat cgctgcattg ctccagacct
     1261 gatcggtatg ggcaaatccg acaaaccaga cctgggttat ttcttcgacg accacgtccg
     1321 cttcatggat gccttcatcg aagccctggg tctggaagag gtcgtcctgg tcattcacga
     1381 ctggggctcc gctctgggtt tccactgggc caagcgcaat ccagagcgcg tcaaaggtat
     1441 tgcatttatg gagttcatcc gccctatccc gacctgggac gaatggccag aatttgcccg
     1501 cgagaccttc caggccttcc gcaccaccga cgtcggccgc aagctgatca tcgatcataa
     1561 cgtttttatc gagggtacgc tgcgtatggg tgtcgtccgc ccgctgactg aagtcgagat
     1621 ggaccattac cgcgagccgt tcctgaatcc tgttgaccgc gagccactgt ggcgcttccc
     1681 aaacgagctg ccaatcgccg gtgagccagc gaacatcgtc gcgctggtcg aagaatacat
     1741 ggactggctg caccagtccc ctgtcccgaa gctgctgttc tggggcaccc caggcgttct
     1801 gatcccaccg gccgaagccg ctcgcctggc caaaagcctg cctaactgca aggctgtgga
     1861 catcggcccg ggtctgaatc tgctgcaaga agacaacccg gacctgatcg gcagcgagat
     1921 cgcgcgctgg ctgtctactc tggagatttc cggtggcgct ccaggattct cctcaatttc
     1981 cgcttccaaa ctgtagtaac cgctcgagtc tagagggccc gtttaaaccc gctgatcagc
     2041 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2101 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2161 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2221 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     2281 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     2341 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2401 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2461 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2521 aaaacttgat tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc
     2581 tctagaaagt ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga
     2641 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga
     2701 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
     2761 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct
     2821 cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc
     2881 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
     2941 gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
     3001 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg
     3061 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
     3121 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg
     3181 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac
     3241 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat
     3301 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag
     3361 gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga
     3421 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
     3481 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
     3541 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg
     3601 ccccagcact cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc
     3661 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     3721 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     3781 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3841 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     3901 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3961 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4021 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4081 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4141 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4201 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4261 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4321 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4381 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4441 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4501 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4561 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4621 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4681 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4741 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4801 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4861 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4921 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4981 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5041 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5101 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5161 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5221 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5281 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5341 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5401 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5461 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5521 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5581 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5641 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5701 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5761 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5821 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5881 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5941 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6001 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6061 atttccccga
//
