LOCUS       sequence-343746-        6088 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Strict cytosolic expression of HaloTag9 in 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_NES_HaloTag9
FEATURES             Location/Qualifiers
     source          1..6088
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(538..1329,1330..1398))
                     /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     1161..1180
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1399..1503)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1578..1597)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1769..2148
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2149..2352
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2303..2323
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2354..2372
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2375..2393
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2403..2427
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2646..3530
                     /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(3574..3591)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3580..3804
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3937..3956)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4088..4135
                     /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             4143..5165
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4143..5165
                     /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    5295..5416
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5332..5351)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5386..5405
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5465..5481)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5465..5481)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5478..5500)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5489..5505
                     /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(5513..5519,5520..5537,5538..5543))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5558..5579
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5696..5713)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5867..6088,1..367))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5947..5966)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
       61 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
      121 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
      181 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
      241 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
      301 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
      361 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
      421 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
      481 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
      541 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
      601 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
      661 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
      721 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
      781 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
      841 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
      901 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
      961 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     1021 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     1081 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     1141 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     1201 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     1261 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     1321 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     1381 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     1441 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     1501 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
     1561 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     1621 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     1681 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
     1741 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
     1801 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     1861 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     1921 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     1981 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     2041 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     2101 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     2161 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     2221 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     2281 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     2341 ataagcagag ctctccctat cagtgataga gatctcccta tcagtgatag agatcgtcga
     2401 cgagctcgtt tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc
     2461 catagaagac accgggaccg atccagcctc cggactctag cgtttaaact taagcttggt
     2521 accgagctcg gatccactag tccagtgtgg tggaattctg cagatatcca gcaaccatgc
     2581 tgcagaacga gcttgctctt aagttggctg gacttgatat taacaagact ggaggttctg
     2641 gatccgaaat cggtactggc tttccattcg acccccatta tgtggaagtc ctgggcgagc
     2701 gcatgcacta cgtcgatgtt ggtccgcgcg atggcacccc tgtgctgttc ctgcacggta
     2761 acccgacctc ctcctacgtg tggcgcaaca tcatcccgca tgttgcaccg acccatcgct
     2821 gcattgctcc agacctgatc ggtatgggca aatccgacaa accagacctg ggttatttct
     2881 tcgacgacca cgtccgcttc atggatgcct tcatcgaagc cctgggtctg gaagaggtcg
     2941 tcctggtcat tcacgactgg ggctccgctc tgggtttcca ctgggccaag cgcaatccag
     3001 agcgcgtcaa aggtattgca tttatggagt tcatccgccc tatcccgacc tgggacgaat
     3061 ggccagaatt tgcccgcgag accttccagg ccttccgcac caccgacgtc ggccgcaagc
     3121 tgatcatcga tcataacgtt tttatcgagg gtacgctgcg tatgggtgtc gtccgcccgc
     3181 tgactgaagt cgagatggac cattaccgcg agccgttcct gaatcctgtt gaccgcgagc
     3241 cactgtggcg cttcccaaac gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc
     3301 tggtcgaaga atacatggac tggctgcacc agtcccctgt cccgaagctg ctgttctggg
     3361 gcaccccagg cgttctgatc ccaccggccg aagccgctcg cctggccaaa agcctgccta
     3421 actgcaaggc tgtggacatc ggcccgggtc tgaatctgct gcaagaagac aacccggacc
     3481 tgatcggcag cgagatcgcg cgctggctgt ctactctgga gatttccggt taaccgctcg
     3541 agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     3601 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3661 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3721 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3781 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     3841 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3901 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     3961 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4021 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     4081 gtacctagaa gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg
     4141 ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca
     4201 gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg
     4261 taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc
     4321 gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg
     4381 gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc
     4441 aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg
     4501 cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa
     4561 tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc
     4621 actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc
     4681 tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct
     4741 ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga
     4801 tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt
     4861 gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc
     4921 ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg
     4981 gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag
     5041 ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct
     5101 gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg
     5161 aatagcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     5221 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     5281 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5341 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5401 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     5461 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5521 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5581 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5641 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5701 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5761 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5821 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5881 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5941 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     6001 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     6061 atagctcacg ctgtaggtat ctcagttc
//
