LOCUS       sequence-343791-        7827 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of HaloTag10 (nuclear) and 9 (mitochondrial surface) 
            in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7827)
  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 7827)
  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_H2B-HaloTag10-T2A-Tomm20-HaloTag9
FEATURES             Location/Qualifiers
     source          1..7827
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(52..71)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        243..622
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        623..826
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     777..797
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    828..846
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    849..867
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     877..901
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1051..1428
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             1441..2325
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDW
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             2341..2394
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(3787..3804)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3793..4017
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4150..4169)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4301..4348
                     /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             4356..5378
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4356..5378
                     /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    5508..5629
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5545..5564)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5599..5618
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5678..5694)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5678..5694)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5691..5713)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5702..5718
                     /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(5726..5732,5733..5750,5751..5756))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5771..5792
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5909..5926)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6080..6668)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6160..6179)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6839..7630,7631..7699))
                     /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     7462..7481
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7700..7804)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
       61 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
      121 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
      181 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
      241 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      301 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      361 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      421 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      481 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      541 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      601 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      661 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      721 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      781 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga
      841 tagagatctc cctatcagtg atagagatcg tcgacgagct cgtttagtga accgtcagat
      901 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
      961 cctccggact ctagcgttta aacttaagct tggtaccgag ctcggatcca ctagtccagt
     1021 gtggtggaat tctgcagata tccagcaacc atgccagagc cagcgaagtc tgctcccgcc
     1081 ccgaaaaagg gctccaagaa ggcggtgact aaggcgcaga agaaaggcgg caagaagcgc
     1141 aagcgcagcc gcaaggagag ctattccatc tatgtgtaca aggttctgaa gcaggtccac
     1201 cctgacaccg gcatttcgtc caaggccatg ggcatcatga attcgtttgt gaacgacatt
     1261 ttcgagcgca tcgcaggtga ggcttcccgc ctggcgcatt acaacaagcg ctcgaccatc
     1321 acctccaggg agatccagac ggccgtgcgc ctgctgctgc ctggggagtt ggccaagcac
     1381 gccgtgtccg agggtactaa ggccatcacc aagtacacca gcgctaaggc tagcggatcc
     1441 gaaatcggta ctggctttcc attcgacccc cattatgtgg aagtcctggg cgagcgcatg
     1501 cactacgtcg atgttggtcc gcgcgatggc acccctgtgc tgttcctgca cggtaacccg
     1561 acctcctcct acgtgtggcg caacatcatc ccgcatgttg caccgaccca tcgctgcatt
     1621 gctccagacc tgatcggtat gggcaaatcc gacaaaccag acctgggtta tttcttcgac
     1681 gaccacgtcc gcttcatgga tgccttcatc gaagccctgg gtctggaaga ggtcgtcctg
     1741 gtcattcacg actggggctc cgctctgggt ttccactggg ccaagcgcaa tccagagcgc
     1801 gtcaaaggta ttgcatttat ggagttcatc cgccctatcc cgacctggga cgaatggcca
     1861 gaatttgccc gcgagacctt ccaggccttc cgcaccaccg acgtcggccg caagctgatc
     1921 atcgattgga acgtttttat cgagggtacg ctgccgatgg gtgtcgtccg cccgctgact
     1981 gaagtcgaga tggaccatta ccgcgagccg ttcctgaatc ctgttgaccg cgagccactg
     2041 tggcgcttcc caaacgagct gccaatcgcc ggtgagccag cgaacatcgt cgcgctggtc
     2101 gaagaataca tggactggct gcaccagtcc cctgtcccga agctgctgtt ctggggcacc
     2161 ccaggcgttc tgatcccacc ggccgaagcc gctcgcctgg ccaaaagcct gcctaactgc
     2221 aaggctgtgg acatcggccc gggtctgaat ctgctgcaag aagacaaccc ggacctgatc
     2281 ggcagcgaga tcgcgcgctg gctgtctact ctggagattt ccggtggccg cggcagtgga
     2341 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccaatggtg
     2401 ggtcggaaca gcgccatcgc cgccggtgta tgcggggccc ttttcattgg gtactgcatc
     2461 tacttcgacc gcaaaagacg aagtgacccc aacttcaaga acaggcttcg agaacgaaga
     2521 aagaaacaga agcttgccaa ggagagagct gggctttcca agttacctga ccttaaagat
     2581 gctgaagctg ttcagaagtt cttccttgaa gaaatacagc ttggtgaaga gttactagct
     2641 caaggtgaat atgagaaggg cgtagaccat ctgacaaatg caattgctgt gtgtggacag
     2701 ccacagcagt tactgcaggt cttacagcaa actcttccac caccagtgtt ccagatgctt
     2761 ctgactaagc tcccaacaat tagtcagaga attgtaagtg ctcagagctt ggctgaagat
     2821 gatgtggaag gcggtagcgg ggatccaccg gtcgccaccg agattggcac agggtttccc
     2881 tttgaccctc attacgttga ggtgctgggt gagaggatgc actatgtcga tgtgggaccc
     2941 cgtgatggca cacctgtgct gtttctccat ggcaatccta ccagctccta tgtgtggagg
     3001 aacatcatac cgcatgtggc tccaacgcac cgatgtatcg caccagatct gatcgggatg
     3061 ggaaagagcg acaaacctga tctgggatac ttcttcgatg accacgtgcg cttcatggac
     3121 gcctttatcg aggctttggg gcttgaggag gtagtgctgg tcatccacga ttggggatct
     3181 gccctggggt ttcattgggc gaaacgcaat cctgagaggg ttaagggcat agccttcatg
     3241 gagttcatac ggcccattcc aacatgggac gaatggccgg aatttgctcg ggaaaccttt
     3301 caagcgttcc gcactaccga tgttgggaga aagctcatca tcgaccacaa cgtgttcatc
     3361 gaaggaactc tccggatggg tgtggtacga cccttgactg aagtcgagat ggaccactat
     3421 agagagccgt ttctcaaccc agtggacaga gaacctctgt ggaggttccc caacgaactt
     3481 cccattgcag gcgaaccagc caatatcgtc gccctcgttg aggagtacat ggactggttg
     3541 catcagtcac cagtacccaa gctgttgttc tggggtacac caggagtgct gattcctccc
     3601 gcagaagcag ctcggctggc taagtctctt cccaactgca aagccgtcga cattggccca
     3661 ggtctgaatc tgcttcagga ggataatcct gatctgattg gcagtgagat tgccagatgg
     3721 ctgtcaaccc tggaaatctc cggtaaccgc tcgagtctag agggcccgtt taaacccgct
     3781 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     3841 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     3901 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     3961 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     4021 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     4081 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     4141 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4201 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4261 accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt
     4321 cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt
     4381 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg
     4441 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg
     4501 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg
     4561 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt
     4621 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg
     4681 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga
     4741 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt
     4801 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg
     4861 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg
     4921 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc
     4981 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg
     5041 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg
     5101 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg
     5161 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc
     5221 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg
     5281 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa
     5341 accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt
     5401 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     5461 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     5521 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5581 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     5641 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5701 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5761 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5821 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5881 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5941 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     6001 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     6061 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     6121 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6181 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     6241 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6301 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6361 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6421 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6481 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6541 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6601 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6661 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6721 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6781 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6841 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6901 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6961 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     7021 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     7081 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     7141 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     7201 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     7261 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7321 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7381 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7441 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7501 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7561 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7621 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7681 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7741 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7801 cgcgcacatt tccccgaaaa gtgccac
//
