LOCUS       sequence-333243-        6484 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HaloTag9 at the mitochondrial surface of mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6484)
  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 6484)
  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_TOMM20_HaloTag9
FEATURES             Location/Qualifiers
     source          1..6484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1508..2392
                     /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(2436..2453)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2442..2666
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2799..2818)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2950..2997
                     /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             3005..4027
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3005..4027
                     /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    4157..4278
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4194..4213)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4248..4267
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4327..4343)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4327..4343)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4340..4362)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4351..4367
                     /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(4375..4381,4382..4399,4400..4405))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4420..4441
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4558..4575)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4729..5317)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4809..4828)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5488..6279,6280..6348))
                     /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     6111..6130
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6349..6453)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga
     1021 attctgcaga tatccagcaa ccatggtggg tcggaacagc gccatcgccg ccggtgtatg
     1081 cggggccctt ttcattgggt actgcatcta cttcgaccgc aaaagacgaa gtgaccccaa
     1141 cttcaagaac aggcttcgag aacgaagaaa gaaacagaag cttgccaagg agagagctgg
     1201 gctttccaag ttacctgacc ttaaagatgc tgaagctgtt cagaagttct tccttgaaga
     1261 aatacagctt ggtgaagagt tactagctca aggtgaatat gagaagggcg tagaccatct
     1321 gacaaatgca attgctgtgt gtggacagcc acagcagtta ctgcaggtct tacagcaaac
     1381 tcttccacca ccagtgttcc agatgcttct gactaagctc ccaacaatta gtcagagaat
     1441 tgtaagtgct cagagcttgg ctgaagatga tgtggaaggc ggtagcgggg atccaccggt
     1501 cgccaccgaa atcggtactg gctttccatt cgacccccat tatgtggaag tcctgggcga
     1561 gcgcatgcac tacgtcgatg ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg
     1621 taacccgacc tcctcctacg tgtggcgcaa catcatcccg catgttgcac cgacccatcg
     1681 ctgcattgct ccagacctga tcggtatggg caaatccgac aaaccagacc tgggttattt
     1741 cttcgacgac cacgtccgct tcatggatgc cttcatcgaa gccctgggtc tggaagaggt
     1801 cgtcctggtc attcacgact ggggctccgc tctgggtttc cactgggcca agcgcaatcc
     1861 agagcgcgtc aaaggtattg catttatgga gttcatccgc cctatcccga cctgggacga
     1921 atggccagaa tttgcccgcg agaccttcca ggccttccgc accaccgacg tcggccgcaa
     1981 gctgatcatc gatcataacg tttttatcga gggtacgctg cgtatgggtg tcgtccgccc
     2041 gctgactgaa gtcgagatgg accattaccg cgagccgttc ctgaatcctg ttgaccgcga
     2101 gccactgtgg cgcttcccaa acgagctgcc aatcgccggt gagccagcga acatcgtcgc
     2161 gctggtcgaa gaatacatgg actggctgca ccagtcccct gtcccgaagc tgctgttctg
     2221 gggcacccca ggcgttctga tcccaccggc cgaagccgct cgcctggcca aaagcctgcc
     2281 taactgcaag gctgtggaca tcggcccggg tctgaatctg ctgcaagaag acaacccgga
     2341 cctgatcggc agcgagatcg cgcgctggct gtccacgctc gagatttccg gctgaccgct
     2401 cgattctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     2461 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     2521 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     2581 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     2641 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     2701 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2761 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2821 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2881 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2941 acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcctt
     3001 ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga
     3061 cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga
     3121 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga
     3181 tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat
     3241 tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt
     3301 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga
     3361 tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg
     3421 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta
     3481 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga
     3541 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg
     3601 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc
     3661 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc
     3721 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc
     3781 gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga
     3841 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg
     3901 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg
     3961 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa
     4021 ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4081 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4141 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4201 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4261 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     4321 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4381 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4441 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4501 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4561 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4621 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4681 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4741 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cggaggtggc gaaacccgac
     4801 aggactataa agataccagg cgtttccccc tggaagctcc ctcatgcgct ctcctgttcc
     4861 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4921 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4981 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5041 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5101 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5161 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5221 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5281 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5341 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5401 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5461 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5521 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5581 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5641 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5701 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5761 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5821 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5881 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5941 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6001 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6061 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6121 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6181 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6241 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6301 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6361 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6421 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6481 cgtc
//
