LOCUS       sequence-347598-        6088 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HaloTag7 at the inner leaflet of the plasma membrane 
            of 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_Lyn11-HaloTag7
FEATURES             Location/Qualifiers
     source          1..6088
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(563..1354,1355..1423))
                     /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     1186..1205
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1424..1528)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1603..1622)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1794..2173
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2174..2377
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2328..2348
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2379..2397
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2400..2418
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2428..2452
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2671..3555
                     /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(3599..3616)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3605..3829
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3962..3981)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4113..4160
                     /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             4168..5190
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4168..5190
                     /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    5320..5441
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5357..5376)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5411..5430
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5490..5506)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5490..5506)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5503..5525)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5514..5530
                     /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(5538..5544,5545..5562,5563..5568))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5583..5604
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5721..5738)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5892..6088,1..392))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5972..5991)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
       61 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
      121 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
      181 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
      241 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
      301 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
      361 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
      421 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
      481 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
      541 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
      601 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
      661 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
      721 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
      781 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
      841 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
      901 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
      961 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     1021 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     1081 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     1141 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     1201 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     1261 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     1321 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     1381 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     1441 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     1501 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     1561 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     1621 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     1681 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
     1741 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
     1801 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     1861 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     1921 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     1981 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     2041 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     2101 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     2161 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     2221 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     2281 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     2341 taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg atagagatct
     2401 ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga tcgcctggag
     2461 acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccggac
     2521 tctagcgttt aaacttaagc ttggtaccga gctcggatcc actagtccag tgtggtggaa
     2581 ttctgcagat atccagcaac catgggatgt ataaaatcaa aagggaaaga cagcgcggga
     2641 gcagatagtg ctggtagtgc tggtggatcc gaaatcggta ctggctttcc attcgacccc
     2701 cattatgtgg aagtcctggg cgagcgcatg cactacgtcg atgttggtcc gcgcgatggc
     2761 acccctgtgc tgttcctgca cggtaacccg acctcctcct acgtgtggcg caacatcatc
     2821 ccgcatgttg caccgaccca tcgctgcatt gctccagacc tgatcggtat gggcaaatcc
     2881 gacaaaccag acctgggtta tttcttcgac gaccacgtcc gcttcatgga tgccttcatc
     2941 gaagccctgg gtctggaaga ggtcgtcctg gtcattcacg actggggctc cgctctgggt
     3001 ttccactggg ccaagcgcaa tccagagcgc gtcaaaggta ttgcatttat ggagttcatc
     3061 cgccctatcc cgacctggga cgaatggcca gaatttgccc gcgagacctt ccaggccttc
     3121 cgcaccaccg acgtcggccg caagctgatc atcgatcaga acgtttttat cgagggtacg
     3181 ctgccgatgg gtgtcgtccg cccgctgact gaagtcgaga tggaccatta ccgcgagccg
     3241 ttcctgaatc ctgttgaccg cgagccactg tggcgcttcc caaacgagct gccaatcgcc
     3301 ggtgagccag cgaacatcgt cgcgctggtc gaagaataca tggactggct gcaccagtcc
     3361 cctgtcccga agctgctgtt ctggggcacc ccaggcgttc tgatcccacc ggccgaagcc
     3421 gctcgcctgg ccaaaagcct gcctaactgc aaggctgtgg acatcggccc gggtctgaat
     3481 ctgctgcaag aagacaaccc ggacctgatc ggcagcgaga tcgcgcgctg gctgtctact
     3541 ctggagattt ccggttaacc gctcgagtct agagggcccg tttaaacccg ctgatcagcc
     3601 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     3661 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     3721 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     3781 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     3841 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     3901 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3961 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     4021 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4081 aaacttgatt agggtgatgg ttcacgtacc tagaagttcc tattccgaag ttcctattct
     4141 ctagaaagta taggaacttc cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag
     4201 aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa
     4261 gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc
     4321 tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc
     4381 ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc
     4441 cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg
     4501 cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg
     4561 ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc
     4621 gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg
     4681 tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg
     4741 cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca
     4801 gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc
     4861 ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg
     4921 catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac
     4981 caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga
     5041 tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga
     5101 agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc
     5161 cccagcactc gtccgagggc aaaggaatag cacgtactac gagatttcga ttccaccgcc
     5221 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     5281 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     5341 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     5401 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     5461 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     5521 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5581 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5641 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5701 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5761 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5821 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5881 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5941 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     6001 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     6061 ccttcgggaa gcgtggcgct ttctcata
//
