LOCUS       sequence-328812-        6412 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nuclear expression of HaloTag9 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6412)
  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 6412)
  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_H2B_HaloTag9
FEATURES             Location/Qualifiers
     source          1..6412
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1043..1420
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             1433..2317
                     /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(2364..2381)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2370..2594
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2727..2746)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2878..2925
                     /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             2933..3955
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2933..3955
                     /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    4085..4206
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4122..4141)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4176..4195
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4255..4271)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4255..4271)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4268..4290)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4279..4295
                     /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(4303..4309,4310..4327,4328..4333))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4348..4369
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4486..4503)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4657..5245)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4737..4756)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5416..6207,6208..6276))
                     /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     6039..6058
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6277..6381)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga
     1021 attctgcaga tatccagcaa ccatgccaga gccagcgaag tctgctcccg ccccgaaaaa
     1081 gggctccaag aaggcggtga ctaaggcgca gaagaaaggc ggcaagaagc gcaagcgcag
     1141 ccgcaaggag agctattcca tctatgtgta caaggttctg aagcaggtcc accctgacac
     1201 cggcatttcg tccaaggcca tgggcatcat gaattcgttt gtgaacgaca ttttcgagcg
     1261 catcgcaggt gaggcttccc gcctggcgca ttacaacaag cgctcgacca tcacctccag
     1321 ggagatccag acggccgtgc gcctgctgct gcctggggag ttggccaagc acgccgtgtc
     1381 cgagggtact aaggccatca ccaagtacac cagcgctaag gctagcggat ccgaaatcgg
     1441 tactggcttt ccattcgacc cccattatgt ggaagtcctg ggcgagcgca tgcactacgt
     1501 cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc cgacctcctc
     1561 ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc catcgctgca ttgctccaga
     1621 cctgatcggt atgggcaaat ccgacaaacc agacctgggt tatttcttcg acgaccacgt
     1681 ccgcttcatg gatgccttca tcgaagccct gggtctggaa gaggtcgtcc tggtcattca
     1741 cgactggggc tccgctctgg gtttccactg ggccaagcgc aatccagagc gcgtcaaagg
     1801 tattgcattt atggagttca tccgccctat cccgacctgg gacgaatggc cagaatttgc
     1861 ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc cgcaagctga tcatcgatca
     1921 taacgttttt atcgagggta cgctgcgtat gggtgtcgtc cgcccgctga ctgaagtcga
     1981 gatggaccat taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt
     2041 cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata
     2101 catggactgg ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt
     2161 tctgatccca ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt
     2221 ggacatcggc ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga
     2281 gatcgcgcgc tggctgtcta ctctggagat ttccggttag taaccgctcg agtctagagg
     2341 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2401 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2461 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2521 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2581 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     2641 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2701 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2761 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2821 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa
     2881 gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc
     2941 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     3001 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     3061 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     3121 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     3181 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     3241 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     3301 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     3361 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     3421 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     3481 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     3541 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     3601 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     3661 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     3721 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     3781 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     3841 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     3901 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt
     3961 actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4021 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4081 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4141 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4201 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     4261 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4321 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4381 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4441 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4501 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4561 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4621 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4681 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4741 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4801 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4861 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4921 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4981 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5041 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     5101 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5161 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5221 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5281 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5341 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5401 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5461 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5521 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5581 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5641 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5701 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5761 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5821 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5881 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5941 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6001 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6061 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6121 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6181 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6241 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6301 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6361 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
