LOCUS       sequence-343741-        6088 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HaloTag9 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-HaloTag9
FEATURES             Location/Qualifiers
     source          1..6088
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        105..484
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        485..688
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     639..659
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    690..708
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    711..729
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     739..763
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             982..1866
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDH
                     NVFIEGTLRMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     primer_bind     complement(1910..1927)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1916..2140
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2273..2292)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2424..2471
                     /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             2479..3501
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2479..3501
                     /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    3631..3752
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3668..3687)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3722..3741
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3801..3817)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3801..3817)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3814..3836)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3825..3841
                     /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(3849..3855,3856..3873,3874..3879))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3894..3915
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4032..4049)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4203..4791)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4283..4302)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4962..5753,5754..5822))
                     /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     5585..5604
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5823..5927)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6002..6021)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
       61 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
      121 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      181 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      241 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      301 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      361 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      421 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      481 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      541 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      601 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      661 acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc tccctatcag
      721 tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc
      781 acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga ctctagcgtt
      841 taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga attctgcaga
      901 tatccagcaa ccatgggatg tataaaatca aaagggaaag acagcgcggg agcagatagt
      961 gctggtagtg ctggtggatc cgaaatcggt actggctttc cattcgaccc ccattatgtg
     1021 gaagtcctgg gcgagcgcat gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg
     1081 ctgttcctgc acggtaaccc gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt
     1141 gcaccgaccc atcgctgcat tgctccagac ctgatcggta tgggcaaatc cgacaaacca
     1201 gacctgggtt atttcttcga cgaccacgtc cgcttcatgg atgccttcat cgaagccctg
     1261 ggtctggaag aggtcgtcct ggtcattcac gactggggct ccgctctggg tttccactgg
     1321 gccaagcgca atccagagcg cgtcaaaggt attgcattta tggagttcat ccgccctatc
     1381 ccgacctggg acgaatggcc agaatttgcc cgcgagacct tccaggcctt ccgcaccacc
     1441 gacgtcggcc gcaagctgat catcgatcat aacgttttta tcgagggtac gctgcgtatg
     1501 ggtgtcgtcc gcccgctgac tgaagtcgag atggaccatt accgcgagcc gttcctgaat
     1561 cctgttgacc gcgagccact gtggcgcttc ccaaacgagc tgccaatcgc cggtgagcca
     1621 gcgaacatcg tcgcgctggt cgaagaatac atggactggc tgcaccagtc ccctgtcccg
     1681 aagctgctgt tctggggcac cccaggcgtt ctgatcccac cggccgaagc cgctcgcctg
     1741 gccaaaagcc tgcctaactg caaggctgtg gacatcggcc cgggtctgaa tctgctgcaa
     1801 gaagacaacc cggacctgat cggcagcgag atcgcgcgct ggctgtctac tctggagatt
     1861 tccggttaac cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg
     1921 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     1981 ggtgccgctc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2041 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2101 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     2161 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2221 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2281 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2341 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2401 tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt
     2461 ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
     2521 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
     2581 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
     2641 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
     2701 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
     2761 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
     2821 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
     2881 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
     2941 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
     3001 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
     3061 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
     3121 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
     3181 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
     3241 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
     3301 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     3361 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     3421 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     3481 cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat
     3541 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     3601 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     3661 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     3721 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     3781 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     3841 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     3901 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3961 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4021 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4081 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4141 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4201 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4261 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4321 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4381 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4441 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4501 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4561 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4621 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4681 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4741 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4801 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4861 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4921 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4981 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5041 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5101 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5161 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5221 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5281 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5341 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5401 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5461 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5521 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5581 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5641 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5701 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5761 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5821 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5881 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5941 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     6001 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     6061 aggtcgctga gtagtgcgcg agcaaaat
//
