LOCUS       sequence-401665-        7310 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tom20 with reversible HaloTagS variant for fluorescent labeling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7310)
  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 7310)
  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-reHaloTagS
FEATURES             Location/Qualifiers
     source          1..7310
                     /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
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLHLLQEDNPDLIG
                     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(3388..3962,3963..3965,3966..3974)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3555..3572)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3782..3801
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3997..4800
                     /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(4060..4079)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4670..4689
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4930..5051
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4967..4986)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5153..5169)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5153..5169)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5166..5188)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5177..5193
                     /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(5201..5207,5208..5225,5226..5231))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5246..5267
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5384..5401)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5555..6143)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5635..5654)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6314..7105,7106..7174))
                     /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     6937..6956
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7175..7279)
                     /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 acctgctgca 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 ctctccctcc ccccccccta acgttactgg
     3421 ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt
     3481 gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc
     3541 taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc
     3601 agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg
     3661 gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc
     3721 tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa
     3781 atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg
     3841 tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa
     3901 aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata
     3961 agcttgccac aacccgggat aattcctgca gccaatatgg gatcggccat tgaacaagat
     4021 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4081 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4141 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     4201 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4261 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4321 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4381 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4441 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4501 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga tgatctcgtc
     4561 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4621 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4681 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4741 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4801 ggggatcaat tctctagata actgatcata atcagccata ccacatttgt agaggtttta
     4861 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     4921 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4981 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5041 aatgtatctt aacgcgtcga gtgcattcta gttgtggttt gtccaaactc atcaatgtat
     5101 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5161 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5221 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5281 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5341 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5401 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5461 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5521 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5581 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5641 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5701 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5761 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5821 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5881 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5941 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6001 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6061 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6121 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6181 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6241 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6301 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6361 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6421 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6481 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6541 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6601 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6661 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6721 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6781 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6841 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6901 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6961 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7021 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7081 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7141 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7201 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7261 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
