LOCUS       sequence-333239-        7231 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HaloTag9 located at the microtubules of mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7231)
  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 7231)
  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_CEP41_HaloTag9
FEATURES             Location/Qualifiers
     source          1..7231
                     /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"
     protein_bind    complement(2181..2205)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2219..2242
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2255..3139
                     /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(3183..3200)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3189..3413
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3546..3565)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3697..3744
                     /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             3752..4774
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3752..4774
                     /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    4904..5025
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4941..4960)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4995..5014
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5074..5090)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5074..5090)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5087..5109)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5098..5114
                     /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(5122..5128,5129..5146,5147..5152))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5167..5188
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5305..5322)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5476..6064)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5556..5575)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6235..7026,7027..7095))
                     /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     6858..6877
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7096..7200)
                     /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 ccatgtccct ccggaggcac attgggaacc ctgagtatct
     1081 gatgaaaagg ataccacaga acccaagata ccagcatatc aaatcaagac tggacactgg
     1141 taacagtatg actaaatata ctgagaagct cgaagagatt aagaaaaatt atagatacaa
     1201 aaaagatgag cttttcaaga gactaaaagt tacaactttt gcccagctga tcatccaagt
     1261 tgcctccctc tctgatcaaa cactggaagt gacagctgag gagattcaaa ggctggaaga
     1321 caatgattct gcagcttcag accctgatgc tgaaaccact gccaggacca atgggaaagg
     1381 aaatccaggt gagcagtcgc cgagccctga gcagttcata aacaacgcag gagcagggga
     1441 ctccagccgc tcaactcttc agagtgtcat cagtggtgtt ggggaactgg atctagacaa
     1501 agggccagtg aagaaagcag agccccatac caaagacaaa ccttatcctg actgcccctt
     1561 cctgctgcta gatgtgcgtg atagagattc ttaccagcag tgccacattg ttggagctta
     1621 cagttaccca attgcaactc tgtctagaac aatgaaccct tattcaaatg atattcttga
     1681 atataaaaat gcccatggca agatcatcat tctgtatgac gatgatgaaa ggctggccag
     1741 tcaggcggcc accaccatgt gcgagcgtgg atttgaaaac ctcttcatgc tttccggagg
     1801 tctaaaagtc ttagctcaga aattcccgga aggactgatt actggttccc tgccagcatc
     1861 ttgccagcag gcccttcctc ctgggtctgc ccggaaacga tccagcccca aagggccacc
     1921 cctaccagct gagaataaat ggagatttac cccagaagac ttaaaaaaga tagaatatta
     1981 tctggaagag gagcaagggc ctgcagatca tcctagccga ctgaaccaag ctaactcctc
     2041 cggaagagag tccaaggtgc ctggtgcccg aagcgctcag aatctgccag gtggcggccc
     2101 cgccagccac tcaaaccccc gctccctcag cagtggtcac ctgcaaggca aaccctggaa
     2161 gtacctcgag tgcggccgca acccagcttt cttgtacaaa gtggtgatgg cagctagcct
     2221 ggaggtgctg ttccagggcc ccttcgaagg atccgaaatc ggtactggct ttccattcga
     2281 cccccattat gtggaagtcc tgggcgagcg catgcactac gtcgatgttg gtccgcgcga
     2341 tggcacccct gtgctgttcc tgcacggtaa cccgacctcc tcctacgtgt ggcgcaacat
     2401 catcccgcat gttgcaccga cccatcgctg cattgctcca gacctgatcg gtatgggcaa
     2461 atccgacaaa ccagacctgg gttatttctt cgacgaccac gtccgcttca tggatgcctt
     2521 catcgaagcc ctgggtctgg aagaggtcgt cctggtcatt cacgactggg gctccgctct
     2581 gggtttccac tgggccaagc gcaatccaga gcgcgtcaaa ggtattgcat ttatggagtt
     2641 catccgccct atcccgacct gggacgaatg gccagaattt gcccgcgaga ccttccaggc
     2701 cttccgcacc accgacgtcg gccgcaagct gatcatcgat cataacgttt ttatcgaggg
     2761 tacgctgcgt atgggtgtcg tccgcccgct gactgaagtc gagatggacc attaccgcga
     2821 gccgttcctg aatcctgttg accgcgagcc actgtggcgc ttcccaaacg agctgccaat
     2881 cgccggtgag ccagcgaaca tcgtcgcgct ggtcgaagaa tacatggact ggctgcacca
     2941 gtcccctgtc ccgaagctgc tgttctgggg caccccaggc gttctgatcc caccggccga
     3001 agccgctcgc ctggccaaaa gcctgcctaa ctgcaaggct gtggacatcg gcccgggtct
     3061 gaatctgctg caagaagaca acccggacct gatcggcagc gagatcgcgc gctggctgtc
     3121 tactctggag atttccggtt aaccgctcga gtctagaggg cccgtttaaa cccgctgatc
     3181 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3241 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3301 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3361 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     3421 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     3481 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3541 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3601 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3661 caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta
     3721 ttctctagaa agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt
     3781 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg
     3841 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
     3901 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc
     3961 gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat
     4021 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt
     4081 tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag
     4141 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat
     4201 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
     4261 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
     4321 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
     4381 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
     4441 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
     4501 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct
     4561 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
     4621 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg
     4681 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
     4741 acgccccagc actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac
     4801 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4861 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     4921 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4981 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     5041 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     5101 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5161 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5221 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5281 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5341 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5401 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5461 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5521 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5581 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5641 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5701 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5761 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5821 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5881 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5941 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6001 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6061 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6121 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6181 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6241 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6301 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6361 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6421 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6481 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6541 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6601 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6661 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6721 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6781 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6841 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6901 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6961 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     7021 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     7081 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7141 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7201 cacatttccc cgaaaagtgc cacctgacgt c
//
