LOCUS       sequence-347602-        6097 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HaloTag9 at the actin filaments of mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6097)
  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 6097)
  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_Lifeact_HaloTag9
FEATURES             Location/Qualifiers
     source          1..6097
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..32)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    21..37
                     /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(45..51,52..69,70..75))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    90..111
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(228..245)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(399..987)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(479..498)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1158..1949,1950..2018))
                     /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     1781..1800
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2019..2123)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2198..2217)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2389..2768
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2769..2972
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2923..2943
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2974..2992
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2995..3013
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3023..3047
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             3197..3247
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     CDS             3275..4159
                     /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(4203..4220)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4209..4433
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4566..4585)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4717..4764
                     /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             4772..5794
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4772..5794
                     /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    5924..6045
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5961..5980)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6015..6034
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(join(6094..6097,1..13))
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(join(6094..6097,1..13))
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
       61 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
      121 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
      181 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
      241 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
      301 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
      361 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
      421 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
      481 agataccagg cgtttcccct tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
      541 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
      601 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
      661 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
      721 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
      781 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
      841 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
      901 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
      961 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     1021 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     1081 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     1141 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     1201 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     1261 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     1321 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     1381 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     1441 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     1501 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     1561 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     1621 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     1681 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     1741 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     1801 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     1861 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     1921 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     1981 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     2041 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     2101 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     2161 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     2221 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     2281 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     2341 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     2401 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     2461 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     2521 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     2581 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     2641 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     2701 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     2761 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     2821 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     2881 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     2941 gtgtacggtg ggaggtctat ataagcagag ctctccctat cagtgataga gatctcccta
     3001 tcagtgatag agatcgtcga cgagctcgtt tagtgaaccg tcagatcgcc tggagacgcc
     3061 atccacgctg ttttgacctc catagaagac accgggaccg atccagcctc cggactctag
     3121 cgtttaaact taagcttggt accgagctcg gatccactag tccagtgtgg tggaattctg
     3181 cagatatcca gcaaccatgg gcgtggccga cctgatcaag aagttcgaga gcatcagcaa
     3241 ggaggagggg gatccaccgg tcgccaccgg atccgaaatc ggtactggct ttccattcga
     3301 cccccattat gtggaagtcc tgggcgagcg catgcactac gtcgatgttg gtccgcgcga
     3361 tggcacccct gtgctgttcc tgcacggtaa cccgacctcc tcctacgtgt ggcgcaacat
     3421 catcccgcat gttgcaccga cccatcgctg cattgctcca gacctgatcg gtatgggcaa
     3481 atccgacaaa ccagacctgg gttatttctt cgacgaccac gtccgcttca tggatgcctt
     3541 catcgaagcc ctgggtctgg aagaggtcgt cctggtcatt cacgactggg gctccgctct
     3601 gggtttccac tgggccaagc gcaatccaga gcgcgtcaaa ggtattgcat ttatggagtt
     3661 catccgccct atcccgacct gggacgaatg gccagaattt gcccgcgaga ccttccaggc
     3721 cttccgcacc accgacgtcg gccgcaagct gatcatcgat cataacgttt ttatcgaggg
     3781 tacgctgcgt atgggtgtcg tccgcccgct gactgaagtc gagatggacc attaccgcga
     3841 gccgttcctg aatcctgttg accgcgagcc actgtggcgc ttcccaaacg agctgccaat
     3901 cgccggtgag ccagcgaaca tcgtcgcgct ggtcgaagaa tacatggact ggctgcacca
     3961 gtcccctgtc ccgaagctgc tgttctgggg caccccaggc gttctgatcc caccggccga
     4021 agccgctcgc ctggccaaaa gcctgcctaa ctgcaaggct gtggacatcg gcccgggtct
     4081 gaatctgctg caagaagaca acccggacct gatcggcagc gagatcgcgc gctggctgtc
     4141 tactctggag atttccggtt aaccgctcga gtctagaggg cccgtttaaa cccgctgatc
     4201 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     4261 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     4321 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     4381 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     4441 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     4501 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4561 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4621 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4681 caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta
     4741 ttctctagaa agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt
     4801 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg
     4861 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
     4921 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc
     4981 gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat
     5041 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt
     5101 tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag
     5161 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat
     5221 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
     5281 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
     5341 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
     5401 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
     5461 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
     5521 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct
     5581 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
     5641 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg
     5701 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
     5761 acgccccagc actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac
     5821 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     5881 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     5941 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     6001 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     6061 gtcgacctct agctagagct tggcgtaatc atggtca
//
