LOCUS       sequence-343793-        6097 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HaloTag7 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_HaloTag7
FEATURES             Location/Qualifiers
     source          1..6097
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(24..41)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(195..783)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(275..294)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(954..1745,1746..1814))
                     /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     1577..1596
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1815..1919)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1994..2013)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2185..2564
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2565..2768
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2719..2739
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2770..2788
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2791..2809
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2819..2843
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2993..3043
                     /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             3071..3955
                     /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(3999..4016)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4005..4229
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4362..4381)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4513..4560
                     /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             4568..5590
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4568..5590
                     /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    5720..5841
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5757..5776)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5811..5830
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5890..5906)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5890..5906)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5903..5925)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5914..5930
                     /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(5938..5944,5945..5962,5963..5968))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5983..6004
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
       61 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
      121 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
      181 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
      241 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccttgga
      301 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
      361 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
      421 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
      481 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
      541 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
      601 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
      661 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
      721 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
      781 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
      841 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
      901 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
      961 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     1021 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     1081 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     1141 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     1201 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     1261 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     1321 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     1381 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     1441 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     1501 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     1561 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     1621 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     1681 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     1741 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     1801 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     1861 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     1921 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
     1981 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
     2041 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
     2101 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
     2161 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
     2221 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     2281 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     2341 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     2401 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     2461 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     2521 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     2581 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     2641 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     2701 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     2761 gcagagctct ccctatcagt gatagagatc tccctatcag tgatagagat cgtcgacgag
     2821 ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata
     2881 gaagacaccg ggaccgatcc agcctccgga ctctagcgtt taaacttaag cttggtaccg
     2941 agctcggatc cactagtcca gtgtggtgga attctgcaga tatccagcaa ccatgggcgt
     3001 ggccgacctg atcaagaagt tcgagagcat cagcaaggag gagggggatc caccggtcgc
     3061 caccggatcc gaaatcggta ctggctttcc attcgacccc cattatgtgg aagtcctggg
     3121 cgagcgcatg cactacgtcg atgttggtcc gcgcgatggc acccctgtgc tgttcctgca
     3181 cggtaacccg acctcctcct acgtgtggcg caacatcatc ccgcatgttg caccgaccca
     3241 tcgctgcatt gctccagacc tgatcggtat gggcaaatcc gacaaaccag acctgggtta
     3301 tttcttcgac gaccacgtcc gcttcatgga tgccttcatc gaagccctgg gtctggaaga
     3361 ggtcgtcctg gtcattcacg actggggctc cgctctgggt ttccactggg ccaagcgcaa
     3421 tccagagcgc gtcaaaggta ttgcatttat ggagttcatc cgccctatcc cgacctggga
     3481 cgaatggcca gaatttgccc gcgagacctt ccaggccttc cgcaccaccg acgtcggccg
     3541 caagctgatc atcgatcaga acgtttttat cgagggtacg ctgccgatgg gtgtcgtccg
     3601 cccgctgact gaagtcgaga tggaccatta ccgcgagccg ttcctgaatc ctgttgaccg
     3661 cgagccactg tggcgcttcc caaacgagct gccaatcgcc ggtgagccag cgaacatcgt
     3721 cgcgctggtc gaagaataca tggactggct gcaccagtcc cctgtcccga agctgctgtt
     3781 ctggggcacc ccaggcgttc tgatcccacc ggccgaagcc gctcgcctgg ccaaaagcct
     3841 gcctaactgc aaggctgtgg acatcggccc gggtctgaat ctgctgcaag aagacaaccc
     3901 ggacctgatc ggcagcgaga tcgcgcgctg gctgtctact ctggagattt ccggttaacc
     3961 gctcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     4021 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     4081 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     4141 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     4201 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     4261 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4321 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4381 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4441 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     4501 ttcacgtacc tagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc
     4561 cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt
     4621 cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt
     4681 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa
     4741 agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga
     4801 cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac
     4861 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat
     4921 ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca
     4981 aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt
     5041 gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga
     5101 tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt
     5161 cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga
     5221 ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt
     5281 ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc
     5341 gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt
     5401 tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc
     5461 cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga
     5521 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc
     5581 aaaggaatag cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     5641 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     5701 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     5761 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     5821 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     5881 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     5941 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     6001 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     6061 ttaatgaatc ggccaacgcg cggggagagg cggtttg
//
