LOCUS       sequence-347603-        7827 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of HaloTag7 (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-HaloTag7-T2A-Tom20-HaloTag9
FEATURES             Location/Qualifiers
     source          1..7827
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             19..396
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             409..1293
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             1309..1362
                     /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(2755..2772)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2761..2985
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3118..3137)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3269..3316
                     /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             3324..4346
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3324..4346
                     /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    4476..4597
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4513..4532)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4567..4586
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4646..4662)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4646..4662)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4659..4681)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4670..4686
                     /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(4694..4700,4701..4718,4719..4724))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4739..4760
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4877..4894)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5048..5636)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5128..5147)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5807..6598,6599..6667))
                     /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     6430..6449
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6668..6772)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6847..6866)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7038..7417
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7418..7621
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7572..7592
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    7623..7641
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7644..7662
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     7672..7696
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
ORIGIN
        1 tgcagatatc cagcaaccat gccagagcca gcgaagtctg ctcccgcccc gaaaaagggc
       61 tccaagaagg cggtgactaa ggcgcagaag aaaggcggca agaagcgcaa gcgcagccgc
      121 aaggagagct attccatcta tgtgtacaag gttctgaagc aggtccaccc tgacaccggc
      181 atttcgtcca aggccatggg catcatgaat tcgtttgtga acgacatttt cgagcgcatc
      241 gcaggtgagg cttcccgcct ggcgcattac aacaagcgct cgaccatcac ctccagggag
      301 atccagacgg ccgtgcgcct gctgctgcct ggggagttgg ccaagcacgc cgtgtccgag
      361 ggtactaagg ccatcaccaa gtacaccagc gctaaggcta gcggatccga aatcggtact
      421 ggctttccat tcgaccccca ttatgtggaa gtcctgggcg agcgcatgca ctacgtcgat
      481 gttggtccgc gcgatggcac ccctgtgctg ttcctgcacg gtaacccgac ctcctcctac
      541 gtgtggcgca acatcatccc gcatgttgca ccgacccatc gctgcattgc tccagacctg
      601 atcggtatgg gcaaatccga caaaccagac ctgggttatt tcttcgacga ccacgtccgc
      661 ttcatggatg ccttcatcga agccctgggt ctggaagagg tcgtcctggt cattcacgac
      721 tggggctccg ctctgggttt ccactgggcc aagcgcaatc cagagcgcgt caaaggtatt
      781 gcatttatgg agttcatccg ccctatcccg acctgggacg aatggccaga atttgcccgc
      841 gagaccttcc aggccttccg caccaccgac gtcggccgca agctgatcat cgatcagaac
      901 gtttttatcg agggtacgct gccgatgggt gtcgtccgcc cgctgactga agtcgagatg
      961 gaccattacc gcgagccgtt cctgaatcct gttgaccgcg agccactgtg gcgcttccca
     1021 aacgagctgc caatcgccgg tgagccagcg aacatcgtcg cgctggtcga agaatacatg
     1081 gactggctgc accagtcccc tgtcccgaag ctgctgttct ggggcacccc aggcgttctg
     1141 atcccaccgg ccgaagccgc tcgcctggcc aaaagcctgc ctaactgcaa ggctgtggac
     1201 atcggcccgg gtctgaatct gctgcaagaa gacaacccgg acctgatcgg cagcgagatc
     1261 gcgcgctggc tgtctactct ggagatttcc ggtggccgcg gcagtggaga gggcagagga
     1321 agtctgctaa catgcggtga cgtcgaggag aatcctggcc caatggtggg tcggaacagc
     1381 gccatcgccg ccggtgtatg cggggccctt ttcattgggt actgcatcta cttcgaccgc
     1441 aaaagacgaa gtgaccccaa cttcaagaac aggcttcgag aacgaagaaa gaaacagaag
     1501 cttgccaagg agagagctgg gctttccaag ttacctgacc ttaaagatgc tgaagctgtt
     1561 cagaagttct tccttgaaga aatacagctt ggtgaagagt tactagctca aggtgaatat
     1621 gagaagggcg tagaccatct gacaaatgca attgctgtgt gtggacagcc acagcagtta
     1681 ctgcaggtct tacagcaaac tcttccacca ccagtgttcc agatgcttct gactaagctc
     1741 ccaacaatta gtcagagaat tgtaagtgct cagagcttgg ctgaagatga tgtggaaggc
     1801 ggtagcgggg atccaccggt cgccaccgag attggcacag ggtttccctt tgaccctcat
     1861 tacgttgagg tgctgggtga gaggatgcac tatgtcgatg tgggaccccg tgatggcaca
     1921 cctgtgctgt ttctccatgg caatcctacc agctcctatg tgtggaggaa catcataccg
     1981 catgtggctc caacgcaccg atgtatcgca ccagatctga tcgggatggg aaagagcgac
     2041 aaacctgatc tgggatactt cttcgatgac cacgtgcgct tcatggacgc ctttatcgag
     2101 gctttggggc ttgaggaggt agtgctggtc atccacgatt ggggatctgc cctggggttt
     2161 cattgggcga aacgcaatcc tgagagggtt aagggcatag ccttcatgga gttcatacgg
     2221 cccattccaa catgggacga atggccggaa tttgctcggg aaacctttca agcgttccgc
     2281 actaccgatg ttgggagaaa gctcatcatc gaccacaacg tgttcatcga aggaactctc
     2341 cggatgggtg tggtacgacc cttgactgaa gtcgagatgg accactatag agagccgttt
     2401 ctcaacccag tggacagaga acctctgtgg aggttcccca acgaacttcc cattgcaggc
     2461 gaaccagcca atatcgtcgc cctcgttgag gagtacatgg actggttgca tcagtcacca
     2521 gtacccaagc tgttgttctg gggtacacca ggagtgctga ttcctcccgc agaagcagct
     2581 cggctggcta agtctcttcc caactgcaaa gccgtcgaca ttggcccagg tctgaatctg
     2641 cttcaggagg ataatcctga tctgattggc agtgagattg ccagatggct gtcaaccctg
     2701 gaaatctccg gtaaccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga
     2761 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     2821 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     2881 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     2941 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     3001 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     3061 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3121 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3181 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3241 ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc tattctctag
     3301 aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
     3361 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
     3421 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
     3481 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
     3541 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     3601 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     3661 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg
     3721 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     3781 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     3841 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     3901 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     3961 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     4021 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     4081 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
     4141 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
     4201 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
     4261 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
     4321 gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc accgccgcct
     4381 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     4441 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     4501 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4561 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     4621 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4681 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4741 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4801 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4861 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4921 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4981 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5041 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5101 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5161 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5221 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5281 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5341 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5401 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5461 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5521 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5581 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5641 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5701 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5761 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5821 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5881 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5941 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6001 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6061 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6121 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6181 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6241 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6301 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6361 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6421 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6481 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6541 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6601 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6661 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6721 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6781 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
     6841 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     6901 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     6961 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     7021 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
     7081 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     7141 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     7201 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     7261 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     7321 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     7381 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     7441 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     7501 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     7561 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     7621 tctccctatc agtgatagag atctccctat cagtgataga gatcgtcgac gagctcgttt
     7681 agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca
     7741 ccgggaccga tccagcctcc ggactctagc gtttaaactt aagcttggta ccgagctcgg
     7801 atccactagt ccagtgtggt ggaattc
//
