LOCUS       sequence-401666-        7309 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tom20 with reversible HaloTagF variant for fluorescent labeling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7309)
  AUTHORS   Holtmannspotter M, Wienbeuker E, Dellmann T, Watrinet I, Garcia-Saez
            AJ, Johnsson K, Kurre R, Piehler J
  TITLE     Reversible Live-Cell Labeling with Retro-engineered HaloTags Enables
            Long-Term High- and Super-Resolution Imaging.
  JOURNAL   Angew Chem Int Ed Engl. 2023 Feb 3:e202219050. doi: 
            10.1002/anie.202219050.
  PUBMED    36735334
REFERENCE   2  (bases 1 to 7309)
  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: "Angew Chem 
            Int Ed Engl. 2023 Feb 3:e202219050. doi: 10.1002/anie.202219050."
COMMENT     Sequence Label: pSEMS-Tom20-mEGFP-reHaloTagF
FEATURES             Location/Qualifiers
     source          1..7309
                     /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"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1362..1364,1365..1367,1368..2078)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1407..1428)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1668..1687)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2015..2036
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2133..3017
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLHYLQEDNPDLIG
                     SEIARWLSTLEISG"
     intron          3086..3315
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     3298..3317
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(3387..3961,3962..3964,3965..3973)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3554..3571)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3781..3800
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3996..4799
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4059..4078)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4669..4688
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4929..5050
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4966..4985)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5152..5168)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5152..5168)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5165..5187)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5176..5192
                     /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(5200..5206,5207..5224,5225..5230))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5245..5266
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5383..5400)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5554..6142)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5634..5653)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6313..7104,7105..7173))
                     /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     6936..6955
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7174..7278)
                     /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 ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat cgatatcatg gtgggtcgga acagcgccat cgccgccggt gtatgcgggg
      961 cccttttcat tgggtactgc atctacttcg accgcaaaag acgaagtgac cccaacttca
     1021 agaacaggct tcgagaacga agaaagaaac agaagcttgc caaggagaga gctgggcttt
     1081 ccaagttacc tgaccttaaa gatgctgaag ctgttcagaa gttcttcctt gaagaaatac
     1141 agcttggtga agagttacta gctcaaggtg aatatgagaa gggcgtagac catctgacaa
     1201 atgcaattgc tgtgtgtgga cagccacagc agttactgca ggtcttacag caaactcttc
     1261 caccaccagt gttccagatg cttctgacta agctcccaac aattagtcag agaattgtaa
     1321 gtgctcagag cttggctgaa gatgatgtgg aagaattcac catggtgagc aagggcgagg
     1381 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     1441 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     1501 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     1561 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     1621 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     1681 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     1741 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     1801 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     1861 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     1921 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtcca
     1981 aactgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     2041 ccgccgggat cactctcggc atggacgagc tgtacaaggg atccctcgag ggccatggca
     2101 caggctcaac aggctcaggc tcatcaggat ccgaaatcgg tactggcttt ccattcgacc
     2161 cccattatgt ggaagtcctg ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg
     2221 gcacccctgt gctgttcctg cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca
     2281 tcccgcatgt tgcaccgacc catcgctgca ttgctccaga cctgatcggt atgggcaaat
     2341 ccgacaaacc agacctgggt tatttcttcg acgaccacgt ccgcttcatg gatgccttca
     2401 tcgaagccct gggtctggaa gaggtcgtcc tggtcattca cgactggggc tccgctctgg
     2461 gtttccactg ggccaagcgc aatccagagc gcgtcaaagg tattgcattt atggagttca
     2521 tccgccctat cccgacctgg gacgaatggc cagaatttgc ccgcgagacc ttccaggcct
     2581 tccgcaccac cgacgtcggc cgcaagctga tcatcgatca gaacgttttt atcgagggta
     2641 cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat taccgcgagc
     2701 cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg
     2761 ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata catggactgg ctgcaccagt
     2821 cccctgtccc gaagctgctg ttctggggca ccccaggcgt tctgatccca ccggccgaag
     2881 ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc ccgggtctgc
     2941 actacctgca agaagacaac ccggacctga tcggcagcga gatcgcgcgc tggctgtcta
     3001 ctctggagat ttccggtgcg gccgcataga taactgatcc agtgtgctgg aattaattcg
     3061 ctgtctgcga gggccagctg ttggggtgag tactccctct caaaagcggg catgacttct
     3121 gcgctaagat tgtcagtttc caaaaacgag gaggatttga tattcacctg gcccgcggtg
     3181 atgcctttga gggtggccgc gtccatctgg tcagaaaaga caatcttttt gttgtcaagc
     3241 ttgaggtgtg gcaggcttga gatctggcca tacacttgag tgacaatgac atccactttg
     3301 cctttctctc cacaggtgtc cactcccagg tccaactgca ggtcgagcat gcatctaggg
     3361 cggccaattc cgcccctctc cccccccccc tctccctccc ccccccctaa cgttactggc
     3421 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg
     3481 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct
     3541 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca
     3601 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg
     3661 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct
     3721 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa
     3781 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt
     3841 atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa
     3901 aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa
     3961 gcttgccaca acccgggata attcctgcag ccaatatggg atcggccatt gaacaagatg
     4021 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4081 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4141 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     4201 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4261 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4321 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4381 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4441 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4501 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg
     4561 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4621 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4681 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4741 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4801 gggatcaatt ctctagataa ctgatcataa tcagccatac cacatttgta gaggttttac
     4861 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     4921 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4981 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5041 atgtatctta acgcgtcgag tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5101 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     5161 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5221 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5281 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5341 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5401 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5461 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5521 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5581 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5641 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5701 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5761 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5821 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5881 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5941 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6001 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6061 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6121 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6181 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6241 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6301 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6361 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6421 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6481 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6541 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6601 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6661 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6721 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6781 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6841 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6901 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6961 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7021 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7081 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7141 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7201 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7261 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
