LOCUS       sequence-343792-        6088 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Strict cytosolic expression of HaloTag7 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6088)
  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 6088)
  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_NES_HaloTag7
FEATURES             Location/Qualifiers
     source          1..6088
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        111..490
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        491..694
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     645..665
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    696..714
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    717..735
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     745..769
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             988..1872
                     /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(1916..1933)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1922..2146
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2279..2298)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2430..2477
                     /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             2485..3507
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2485..3507
                     /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    3637..3758
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3674..3693)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3728..3747
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3807..3823)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3807..3823)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3820..3842)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3831..3847
                     /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(3855..3861,3862..3879,3880..3885))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3900..3921
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4038..4055)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4209..4797)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4289..4308)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4968..5759,5760..5828))
                     /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     5591..5610
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5829..5933)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6008..6027)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
       61 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
      121 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
      181 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      241 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      301 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      361 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      421 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      481 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      541 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      601 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      661 gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata gagatctccc
      721 tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg
      781 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccggactct
      841 agcgtttaaa cttaagcttg gtaccgagct cggatccact agtccagtgt ggtggaattc
      901 tgcagatatc cagcaaccat gctgcagaac gagcttgctc ttaagttggc tggacttgat
      961 attaacaaga ctggaggttc tggatccgaa atcggtactg gctttccatt cgacccccat
     1021 tatgtggaag tcctgggcga gcgcatgcac tacgtcgatg ttggtccgcg cgatggcacc
     1081 cctgtgctgt tcctgcacgg taacccgacc tcctcctacg tgtggcgcaa catcatcccg
     1141 catgttgcac cgacccatcg ctgcattgct ccagacctga tcggtatggg caaatccgac
     1201 aaaccagacc tgggttattt cttcgacgac cacgtccgct tcatggatgc cttcatcgaa
     1261 gccctgggtc tggaagaggt cgtcctggtc attcacgact ggggctccgc tctgggtttc
     1321 cactgggcca agcgcaatcc agagcgcgtc aaaggtattg catttatgga gttcatccgc
     1381 cctatcccga cctgggacga atggccagaa tttgcccgcg agaccttcca ggccttccgc
     1441 accaccgacg tcggccgcaa gctgatcatc gatcagaacg tttttatcga gggtacgctg
     1501 ccgatgggtg tcgtccgccc gctgactgaa gtcgagatgg accattaccg cgagccgttc
     1561 ctgaatcctg ttgaccgcga gccactgtgg cgcttcccaa acgagctgcc aatcgccggt
     1621 gagccagcga acatcgtcgc gctggtcgaa gaatacatgg actggctgca ccagtcccct
     1681 gtcccgaagc tgctgttctg gggcacccca ggcgttctga tcccaccggc cgaagccgct
     1741 cgcctggcca aaagcctgcc taactgcaag gctgtggaca tcggcccggg tctgaatctg
     1801 ctgcaagaag acaacccgga cctgatcggc agcgagatcg cgcgctggct gtctactctg
     1861 gagatttccg gttaaccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg
     1921 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     1981 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     2041 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2101 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     2161 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     2221 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2281 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2341 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2401 cttgattagg gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta
     2461 gaaagtatag gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag
     2521 tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa
     2581 tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc
     2641 gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg
     2701 attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc
     2761 cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag
     2821 ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc
     2881 ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg
     2941 attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc
     3001 gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac
     3061 ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg
     3121 gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc
     3181 ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat
     3241 ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa
     3301 ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc
     3361 gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc
     3421 gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc
     3481 agcactcgtc cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc
     3541 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     3601 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     3661 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     3721 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     3781 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3841 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3901 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3961 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4021 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4081 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4141 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4201 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4261 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4321 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4381 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4441 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4501 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4561 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4621 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4681 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4741 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4801 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4861 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4921 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4981 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5041 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5101 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5161 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5221 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5281 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5341 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5401 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5461 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5521 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5581 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5641 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5701 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5761 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5821 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5881 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5941 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
     6001 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     6061 tgttggaggt cgctgagtag tgcgcgag
//
