LOCUS       sequence-347597-        6070 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Peroxysomal expression of HaloTag7 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_HaloTag7_SKL
FEATURES             Location/Qualifiers
     source          1..6070
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(116..135)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        307..686
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        687..890
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     841..861
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    892..910
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    913..931
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     941..965
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1124..2008
                     /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(2094..2111)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2100..2324
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2457..2476)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2608..2655
                     /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             2663..3685
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2663..3685
                     /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    3815..3936
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3852..3871)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3906..3925
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3985..4001)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3985..4001)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3998..4020)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4009..4025
                     /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(4033..4039,4040..4057,4058..4063))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4078..4099
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4216..4233)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4387..4975)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4467..4486)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5146..5937,5938..6006))
                     /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     5769..5788
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6007..6070,1..41))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
       61 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
      121 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      181 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
      241 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      301 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      361 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      421 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      481 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      541 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      601 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      661 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      721 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      781 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      841 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca
      901 gtgatagaga tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc
      961 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
     1021 ccagcctccg gactctagcg tttaaactta agcttggtac cgagctcgga tccactagtc
     1081 cagtgtggtg gaattctgca gatatccagc aaccatggga tccgaaatcg gtactggctt
     1141 tccattcgac ccccattatg tggaagtcct gggcgagcgc atgcactacg tcgatgttgg
     1201 tccgcgcgat ggcacccctg tgctgttcct gcacggtaac ccgacctcct cctacgtgtg
     1261 gcgcaacatc atcccgcatg ttgcaccgac ccatcgctgc attgctccag acctgatcgg
     1321 tatgggcaaa tccgacaaac cagacctggg ttatttcttc gacgaccacg tccgcttcat
     1381 ggatgccttc atcgaagccc tgggtctgga agaggtcgtc ctggtcattc acgactgggg
     1441 ctccgctctg ggtttccact gggccaagcg caatccagag cgcgtcaaag gtattgcatt
     1501 tatggagttc atccgcccta tcccgacctg ggacgaatgg ccagaatttg cccgcgagac
     1561 cttccaggcc ttccgcacca ccgacgtcgg ccgcaagctg atcatcgatc agaacgtttt
     1621 tatcgagggt acgctgccga tgggtgtcgt ccgcccgctg actgaagtcg agatggacca
     1681 ttaccgcgag ccgttcctga atcctgttga ccgcgagcca ctgtggcgct tcccaaacga
     1741 gctgccaatc gccggtgagc cagcgaacat cgtcgcgctg gtcgaagaat acatggactg
     1801 gctgcaccag tcccctgtcc cgaagctgct gttctggggc accccaggcg ttctgatccc
     1861 accggccgaa gccgctcgcc tggccaaaag cctgcctaac tgcaaggctg tggacatcgg
     1921 cccgggtctg aatctgctgc aagaagacaa cccggacctg atcggcagcg agatcgcgcg
     1981 ctggctgtct actctggaga tttccggtgg cgctccagga ttctcctcaa tttccgcttc
     2041 caaactgtag taaccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     2101 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2161 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2221 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2281 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     2341 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     2401 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2461 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2521 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2581 tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga
     2641 aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt
     2701 tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc
     2761 tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc
     2821 cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat
     2881 tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg
     2941 tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc
     3001 ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg
     3061 cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat
     3121 tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt
     3181 cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct
     3241 cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt
     3301 cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt
     3361 ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc
     3421 ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact
     3481 ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga
     3541 cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc
     3601 ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag
     3661 cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca ccgccgcctt
     3721 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     3781 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     3841 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3901 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     3961 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4021 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4081 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4141 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4201 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4261 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4321 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4381 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4441 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4501 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4561 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4621 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4681 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4741 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4801 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     4861 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4921 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4981 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5041 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5101 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5161 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5221 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5281 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5341 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5401 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5461 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5521 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5581 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5641 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5701 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5761 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5821 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5881 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5941 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6001 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6061 cggatacata
//
