LOCUS       sequence-343745-        7819 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HaloTag9 at the nuclear lamina of the mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7819)
  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 7819)
  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_HaloTag9_LamB1
FEATURES             Location/Qualifiers
     source          1..7819
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(114..133)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        305..684
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        685..888
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     839..859
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    890..908
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    911..929
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     939..963
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1122..2006
                     /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(3841..3858)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3847..4071
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4204..4223)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4355..4402
                     /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             4410..5432
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4410..5432
                     /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    5562..5683
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5599..5618)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5653..5672
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5732..5748)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5732..5748)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5745..5767)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5756..5772
                     /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(5780..5786,5787..5804,5805..5810))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5825..5846
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5963..5980)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6134..6722)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6214..6233)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6893..7684,7685..7753))
                     /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     7516..7535
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7754..7819,1..39))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
       61 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
      121 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
      181 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
      241 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
      301 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      361 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      421 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      481 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      541 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      601 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      661 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      721 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      781 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      841 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt
      901 gatagagatc tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag
      961 atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc
     1021 agcctccgga ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca
     1081 gtgtggtgga attctgcaga tatccagcaa ccatgggatc cgaaatcggt actggctttc
     1141 cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat gcactacgtc gatgttggtc
     1201 cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc gacctcctcc tacgtgtggc
     1261 gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat tgctccagac ctgatcggta
     1321 tgggcaaatc cgacaaacca gacctgggtt atttcttcga cgaccacgtc cgcttcatgg
     1381 atgccttcat cgaagccctg ggtctggaag aggtcgtcct ggtcattcac gactggggct
     1441 ccgctctggg tttccactgg gccaagcgca atccagagcg cgtcaaaggt attgcattta
     1501 tggagttcat ccgccctatc ccgacctggg acgaatggcc agaatttgcc cgcgagacct
     1561 tccaggcctt ccgcaccacc gacgtcggcc gcaagctgat catcgatcat aacgttttta
     1621 tcgagggtac gctgcgtatg ggtgtcgtcc gcccgctgac tgaagtcgag atggaccatt
     1681 accgcgagcc gttcctgaat cctgttgacc gcgagccact gtggcgcttc ccaaacgagc
     1741 tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt cgaagaatac atggactggc
     1801 tgcaccagtc ccctgtcccg aagctgctgt tctggggcac cccaggcgtt ctgatcccac
     1861 cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg caaggctgtg gacatcggcc
     1921 cgggtctgaa tctgctgcaa gaagacaacc cggacctgat cggcagcgag atcgcgcgct
     1981 ggctgtctac tctggagatt tccggttccg gactcagatc tcgagctcaa gcttcgatgg
     2041 cgactgcgac ccccgtgccg ccgcggatgg gcagccgcgc tggcggcccc accacgccgc
     2101 tgagccccac gcgcctgtcg cggctccagg agaaggagga gctgcgcgag ctcaatgacc
     2161 ggctggcggt gtacatcgac aaggtgcgca gcctggagac ggagaacagc gcgctgcagc
     2221 tgcaggtgac ggagcgcgag gaggtgcgcg gccgtgagct caccggcctc aaggcgctct
     2281 acgagaccga gctggccgac gcgcgacgcg cgctcgacga cacggcccgc gagcgcgcca
     2341 agctgcagat cgagctgggc aagtgcaagg cggaacacga ccagctgctc ctcaactatg
     2401 ctaagaagga atctgatctt aatggcgccc agatcaagct tcgagaatat gaagcagcac
     2461 tcaattcgaa agatgcagct cttgctactg cacttggtga caaaaaaagt ttagagggag
     2521 atttggagga tctgaaggat cagattgccc agttggaagc ctccttagct gcagccaaaa
     2581 aacagttagc agatgaaact ttacttaaag tagatttgga gaatcgttgt cagagcctta
     2641 ctgaggactt ggagtttcgc aaaagcatgt atgaagagga gattaacgag accagaagga
     2701 agcatgaaac gcgcttggta gaggtggatt ctgggcgtca aattgagtat gagtacaagc
     2761 tggcgcaagc ccttcatgag atgagagagc aacatgatgc ccaagtgagg ctgtataagg
     2821 aggagctgga gcagacttac catgccaaac ttgagaatgc cagactgtca tcagagatga
     2881 atacttctac tgtcaacagt gccagggaag aactgatgga aagccgcatg agaattgaga
     2941 gcctttcatc ccagctttct aatctacaga aagagtctag agcatgtttg gaaaggattc
     3001 aagaattaga ggacttgctt gctaaagaaa aagacaactc tcgtcgcatg ctgacagaca
     3061 aagagagaga gatggcggaa ataagggatc aaatgcagca acagctgaat gactatgaac
     3121 agcttcttga tgtaaagtta gccctggaca tggaaatcag tgcttacagg aaactcttag
     3181 aaggcgaaga agagaggttg aagctgtctc caagcccttc ttcccgtgtg acagtatccc
     3241 gagcatcctc aagtcgtagt gtacgtacaa ctagaggaaa gcggaagagg gttgatgtgg
     3301 aagaatcaga ggcgagtagt agtgttagca tctctcattc cgcctcagcc actggaaatg
     3361 tttgcatcga agaaattgat gttgatggga aatttatccg cttgaagaac acttctgaac
     3421 aggatcaacc aatgggaggc tgggagatga tcagaaaaat tggagacaca tcagtcagtt
     3481 ataaatatac ctcaagatat gtgctgaagg caggccagac tgttacaatt tgggctgcaa
     3541 acgctggtgt cacagccagc cccccaactg acctcatctg gaagaaccag aactcgtggg
     3601 gcactggcga agatgtgaag gttatattga aaaattctca gggagaggag gttgctcaaa
     3661 gaagtacagt ctttaaaaca accatacctg aagaagagga ggaggaggaa gaagcagctg
     3721 gagtggttgt tgaggaagaa cttttccacc agcagggaac cccaagagca tccaatagaa
     3781 gctgtgcaat tatgtagtaa ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag
     3841 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3901 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3961 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     4021 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     4081 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     4141 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     4201 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4261 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4321 aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt
     4381 ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg
     4441 agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg
     4501 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata
     4561 gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc
     4621 tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct
     4681 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc
     4741 tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg
     4801 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat
     4861 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg
     4921 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc
     4981 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa
     5041 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca
     5101 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga
     5161 ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg
     5221 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc
     5281 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca
     5341 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac
     5401 gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg
     5461 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     5521 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     5581 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     5641 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     5701 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     5761 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     5821 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5881 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5941 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     6001 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     6061 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     6121 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     6181 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     6241 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     6301 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     6361 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6421 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6481 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6541 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6601 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     6661 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6721 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6781 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6841 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6901 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6961 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     7021 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     7081 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     7141 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     7201 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     7261 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     7321 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     7381 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7441 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7501 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7561 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7621 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7681 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7741 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7801 tcatgagcgg atacatatt
//
