LOCUS       sequence-347600-        6217 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HaloTag9 at the inner membrane of mitochondria in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6217)
  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 6217)
  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_[Cox8a]x2_HaloTag9
FEATURES             Location/Qualifiers
     source          1..6217
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(109..128)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        300..679
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        680..883
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     834..854
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    885..903
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    906..924
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     934..958
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1111..1185
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1198..1269
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1306..2190
                     /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(2234..2251)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2240..2464
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2597..2616)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2748..2795
                     /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             2803..3825
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2803..3825
                     /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    3955..4076
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3992..4011)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4046..4065
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4125..4141)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4125..4141)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4138..4160)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4149..4165
                     /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(4173..4179,4180..4197,4198..4203))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4218..4239
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4356..4373)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4527..5115)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4607..4626)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5286..6077,6078..6146))
                     /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     5909..5928
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6147..6217,1..34))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
       61 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
      121 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
      181 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
      241 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
      301 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      361 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      421 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      481 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      541 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      601 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      661 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      721 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      781 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      841 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctcccta tcagtgatag
      901 agatctccct atcagtgata gagatcgtcg acgagctcgt ttagtgaacc gtcagatcgc
      961 ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct
     1021 ccggactcta gcgtttaaac ttaagcttgg taccgagctc ggatccacta gtccagtgtg
     1081 gtggaattct gcagatatcc agcaaccacc atgtccgtcc tgacgccgct gctgctgcgg
     1141 ggcttgacag gctcggcccg gcggctccca gtgccgcgcg ccaagatcca ttcgttgtcc
     1201 gtcctgacgc cgctgctgct gcggggcttg acaggctcgg cccggcggct cccagtgccg
     1261 cgcgccaaga tccattcgtt gggaggcagc ggaggcagcg gatccgaaat cggtactggc
     1321 tttccattcg acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt
     1381 ggtccgcgcg atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg
     1441 tggcgcaaca tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc
     1501 ggtatgggca aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc
     1561 atggatgcct tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg
     1621 ggctccgctc tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca
     1681 tttatggagt tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcgag
     1741 accttccagg ccttccgcac caccgacgtc ggccgcaagc tgatcatcga tcataacgtt
     1801 tttatcgagg gtacgctgcg tatgggtgtc gtccgcccgc tgactgaagt cgagatggac
     1861 cattaccgcg agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac
     1921 gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac
     1981 tggctgcacc agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc
     2041 ccaccggccg aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc
     2101 ggcccgggtc tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg
     2161 cgctggctgt ctactctgga gatttccggt taaccgctcg agtctagagg gcccgtttaa
     2221 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2281 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2341 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     2401 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     2461 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     2521 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2581 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2641 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2701 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc
     2761 cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc
     2821 gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag
     2881 ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc
     2941 ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt
     3001 gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg
     3061 acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa
     3121 ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt
     3181 agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg
     3241 cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac
     3301 gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac
     3361 tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac
     3421 aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac
     3481 gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc
     3541 tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc
     3601 cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct
     3661 tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca
     3721 caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat
     3781 agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat
     3841 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     3901 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     3961 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4021 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4081 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4141 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4201 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4261 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4321 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4381 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4441 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4501 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4561 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4621 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4681 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4741 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4801 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4861 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4921 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4981 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5041 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5101 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5161 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5221 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5281 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5341 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5401 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5461 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5521 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5581 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5641 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5701 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5761 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5821 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5881 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5941 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6001 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6061 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6121 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6181 cagggttatt gtctcatgag cggatacata tttgaat
//
