LOCUS       sequence-347601-        6328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HaloTag9 at the outer leaflet of the plasma membrane 
            of mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6328)
  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 6328)
  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_IgKchL_HA_HaloTag9_myc_PDGFRtmb
FEATURES             Location/Qualifiers
     source          1..6328
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(102..121)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        293..672
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        673..876
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     827..847
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    878..896
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    899..917
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     927..951
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     sig_peptide     1101..1160
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             1164..1190
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1218..2102
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDH
                     NVFIEGTLRMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             2112..2141
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2145..2291
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     primer_bind     complement(2338..2355)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2344..2568
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2701..2720)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2852..2899
                     /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             2907..3929
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2907..3929
                     /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    4059..4180
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4096..4115)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4150..4169
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4229..4245)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4229..4245)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4242..4264)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4253..4269
                     /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(4277..4283,4284..4301,4302..4307))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4322..4343
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4460..4477)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4631..5219)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4711..4730)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5390..6181,6182..6250))
                     /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     6013..6032
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6251..6328,1..27))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
       61 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
      121 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
      181 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
      241 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga
      301 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
      361 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
      421 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      481 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      541 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
      601 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      661 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      721 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      781 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
      841 aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga tagagatctc
      901 cctatcagtg atagagatcg tcgacgagct cgtttagtga accgtcagat cgcctggaga
      961 cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccggact
     1021 ctagcgttta aacttaagct tggtaccgag ctcggatcca ctagtccagt gtggtggaat
     1081 tctgcagata tccagcaacc atggagacag acacactcct gctatgggta ctgctgctct
     1141 gggttccagg ttccactggt gactatccat atgatgttcc agattatgct ggggcccagc
     1201 cggccagatc aggatccgaa atcggtactg gctttccatt cgacccccat tatgtggaag
     1261 tcctgggcga gcgcatgcac tacgtcgatg ttggtccgcg cgatggcacc cctgtgctgt
     1321 tcctgcacgg taacccgacc tcctcctacg tgtggcgcaa catcatcccg catgttgcac
     1381 cgacccatcg ctgcattgct ccagacctga tcggtatggg caaatccgac aaaccagacc
     1441 tgggttattt cttcgacgac cacgtccgct tcatggatgc cttcatcgaa gccctgggtc
     1501 tggaagaggt cgtcctggtc attcacgact ggggctccgc tctgggtttc cactgggcca
     1561 agcgcaatcc agagcgcgtc aaaggtattg catttatgga gttcatccgc cctatcccga
     1621 cctgggacga atggccagaa tttgcccgcg agaccttcca ggccttccgc accaccgacg
     1681 tcggccgcaa gctgatcatc gatcataacg tttttatcga gggtacgctg cgtatgggtg
     1741 tcgtccgccc gctgactgaa gtcgagatgg accattaccg cgagccgttc ctgaatcctg
     1801 ttgaccgcga gccactgtgg cgcttcccaa acgagctgcc aatcgccggt gagccagcga
     1861 acatcgtcgc gctggtcgaa gaatacatgg actggctgca ccagtcccct gtcccgaagc
     1921 tgctgttctg gggcacccca ggcgttctga tcccaccggc cgaagccgct cgcctggcca
     1981 aaagcctgcc taactgcaag gctgtggaca tcggcccggg tctgaatctg ctgcaagaag
     2041 acaacccgga cctgatcggc agcgagatcg cgcgctggct gtctactctg gagatttccg
     2101 gtggcgtcga cgaacaaaaa ctcatctcag aagaggatct gaatgctgtg ggccaggaca
     2161 cgcaggaggt catcgtggtg ccacactcct tgccctttaa ggtggtggtg atctcagcca
     2221 tcctggccct ggtggtgctc accatcatct cccttatcat cctcatcatg ctttggcaga
     2281 agaagccacg ttagtaaccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct
     2341 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     2401 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     2461 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     2521 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     2581 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     2641 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2701 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2761 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2821 aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc
     2881 tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga
     2941 agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
     3001 aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
     3061 gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc
     3121 cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc
     3181 gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc
     3241 agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt
     3301 tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
     3361 cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt
     3421 ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc
     3481 acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag
     3541 cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct
     3601 tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc
     3661 atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
     3721 aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat
     3781 gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa
     3841 gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc
     3901 ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg
     3961 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4021 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4081 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4141 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     4201 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4261 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4321 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4381 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4441 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4501 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4561 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4621 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4681 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4741 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4801 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4861 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4921 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4981 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5041 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5101 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5161 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5221 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5281 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5341 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5401 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5461 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5521 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5581 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5641 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5701 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5761 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5821 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5881 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5941 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6001 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6061 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6121 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6181 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6241 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6301 tgagcggata catatttgaa tgtattta
//
