LOCUS       sequence-374609-        7906 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For C-terminal -Halo Tagging of a gene of interest with TRP1 
            selection marker in yeast S. cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7906)
  AUTHORS   Podh NK, Das A, Dey P, Paliwal S, Mehta G
  TITLE     Single-molecule tracking for studying protein dynamics and 
            target-search mechanism in live cells of S. cerevisiae.
  JOURNAL   STAR Protoc. 2022 Dec 16;3(4):101900. doi: 
            10.1016/j.xpro.2022.101900. Epub 2022 Dec 5.
  PUBMED    36595957
REFERENCE   2  (bases 1 to 7906)
  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: "STAR 
            Protoc."; date: "2022-12-16"; volume: "3(4):101900. doi"; pages: " 
            10.1016/j.xpro.2022.101900. Epub 2022 Dec 5"
COMMENT     Sequence Label: pTSK573
FEATURES             Location/Qualifiers
     source          1..7906
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             52..936
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     promoter        1068..1345
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             1346..2020
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     primer_bind     complement(2297..2316)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2298..2316)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2323..2340)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2323..2339)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2332..2354)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2480..2935
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2567..2586)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2777..2798
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3195..3857)
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     promoter        complement(3858..4045)
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     primer_bind     complement(4192..4211)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4311..4333
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4371..4389)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4457..4561
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4562..4630,4631..5422)
                     /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     complement(4780..4799)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5593..6181
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6082..6101
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6335..6352
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6469..6490
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6505..6510,6511..6528,6529..6535)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6543..6559
                     /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)."
     primer_bind     6548..6570
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6567..6583
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6567..6583
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        6604..6622
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 caagcggccg ccgctgctgc cgctgcagct gctgcagctg cgatgggttc cgaaatcggt
       61 actggctttc cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat gcactacgtc
      121 gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc gacctcctcc
      181 tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat tgctccagac
      241 ctgatcggta tgggcaaatc cgacaaacca gacctgggtt atttcttcga cgaccacgtc
      301 cgcttcatgg atgccttcat cgaagccctg ggtctggaag aggtcgtcct ggtcattcac
      361 gactggggct ccgctctggg tttccactgg gccaagcgca atccagagcg cgtcaaaggt
      421 attgcattta tggagttcat ccgccctatc ccgacctggg acgaatggcc agaatttgcc
      481 cgcgagacct tccaggcctt ccgcaccacc gacgtcggcc gcaagctgat catcgatcag
      541 aacgttttta tcgagggtac gctgccgatg ggtgtcgtcc gcccgctgac tgaagtcgag
      601 atggaccatt accgcgagcc gttcctgaat cctgttgacc gcgagccact gtggcgcttc
      661 ccaaacgagc tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt cgaagaatac
      721 atggactggc tgcaccagtc ccctgtcccg aagctgctgt tctggggcac cccaggcgtt
      781 ctgatcccac cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg caaggctgtg
      841 gacatcggcc cgggtctgaa tctgctgcaa gaagacaacc cggacctgat cggcagcgag
      901 atcgcgcgct ggctgtctac tctggagatt tccggttaac cgcggaaacg gcagccccga
      961 tctaaaagag ctgacaggga aatggtcaga aaaagaaacg tgcacccgcc cgtctggacg
     1021 cgccgctcac ccgcacggca gagaccaatc agtaaaaatc aacggttaac gacattacta
     1081 tatatataat ataggaagca tttaatagaa cagcatcgta atatatgtgt actttgcagt
     1141 tatgacgcca gatggcagta gtggaagata ttctttattg aaaaatagct tgtcacctta
     1201 cgtacaatct tgatccggag cttttctttt tttgccgatt aagaattcgg tcgaaaaaag
     1261 aaaaggagag ggccaagagg gagggcattg gtgactattg agcacgtgag tatacgtgat
     1321 taagcacaca aaggcagctt ggagtatgtc tgttattaat ttcacaggta gttctggtcc
     1381 attggtgaaa gtttgcggct tgcagagcac agaggccgca gaatgtgctc tagattccga
     1441 tgctgacttg ctgggtatta tatgtgtgcc caatagaaag agaacaattg acccggttat
     1501 tgcaaggaaa atttcaagtc ttgtaaaagc atataaaaat agttcaggca ctccgaaata
     1561 cttggttggc gtgtttcgta atcaacctaa ggaggatgtt ttggctctgg tcaatgatta
     1621 cggcattgat atcgtccaac tgcatggaga tgagtcgtgg caagaatacc aagagttcct
     1681 cggtttgcca gttattaaaa gactcgtatt tccaaaagac tgcaacatac tactcagtgc
     1741 agcttcacag aaacctcatt cgtttattcc cttgtttgat tcagaagcag gtgggacagg
     1801 tgaacttttg gattggaact cgatttctga ctgggttgga aggcaagaga gccccgaaag
     1861 cttacatttt atgttagctg gtggactgac gccagaaaat gttggtgatg cgcttagatt
     1921 aaatggcgtt attggtgttg atgtaagcgg aggtgtggag acaaatggtg taaaagactc
     1981 taacaaaata gcaaatttcg tcaaaaatgc taagaaatag gagctctgca tctggacatc
     2041 tatataaata tacatataca tatattggcg gttgtttgtt ctcccggcaa gctggcagtc
     2101 gggccgagat tcggataaat ttttccattc gtatacatat acatagatat atacaattat
     2161 aaaatttgtc caaggctgca caggatacgt aaccatccta aagttttagg ttttttaact
     2221 ttttttctgt ctcttcaatt gtcgaattgc ttattctatc ctttctggag ttcgtattta
     2281 aatgagctcc aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca
     2341 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     2401 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     2461 cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2521 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2581 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2641 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2701 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2761 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     2821 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     2881 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttcctga
     2941 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatagat ccgtcgagtt
     3001 caagagaaaa aaaaagaaaa agcaaaaaga aaaaaggaaa gcgcgcctcg ttcagaatga
     3061 cacgtataga atgatgcatt accttgtcat cttcagtatc atactgttcg tatacatact
     3121 tactgacatt cataggtata catatataca catgtatata tatcgtatgc tgcagcttta
     3181 aataatcggt gtcactacat aagaacacct ttggtggagg gaacatcgtt ggtaccattg
     3241 ggcgaggtgg cttctcttat ggcaaccgca agagccttga acgcactctc actacggtga
     3301 tgatcattct tgcctcgcag acaatcaacg tggagggtaa ttctgctagc ctctgcaaag
     3361 ctttcaagaa aatgcgggat catctcgcaa gagagatctc ctactttctc cctttgcaaa
     3421 ccaagttcga caactgcgta cggcctgttc gaaagatcta ccaccgctct ggaaagtgcc
     3481 tcatccaaag gcgcaaatcc tgatccaaac ctttttactc cacgcacggc ccctagggcc
     3541 tctttaaaag cttgaccgag agcaatcccg cagtcttcag tggtgtgatg gtcgtctatg
     3601 tgtaagtcac caatgcactc aacgattagc gaccagccgg aatgcttggc cagagcatgt
     3661 atcatatggt ccagaaaccc tatacctgtg tggacgttaa tcacttgcga ttgtgtggcc
     3721 tgttctgcta ctgcttctgc ctctttttct gggaagatcg agtgctctat cgctagggga
     3781 ccacccttta aagagatcgc aatctgaatc ttggtttcat ttgtaatacg ctttactagg
     3841 gctttctgct ctgtcatctt tgccttcgtt tatcttgcct gctcattttt tagtatattc
     3901 ttcgaagaaa tcacattact ttatataatg tataattcat tatgtgataa tgccaatcgc
     3961 taagaaaaaa aaagagtcat ccgctaggtg gaaaaaaaaa aatgaaaatc attaccgagg
     4021 cataaaaaaa tatagagtgt actagaggag gccaagagta atagaaaaag aaaattgcgg
     4081 gaaaggactg tgttatgact tccctgacta atgccgtgtt caaacgatac ctggcagtga
     4141 ctcctagcgc tcaccaagct cttaaaacgg gaatttatgg tgcactctca gtacaatctg
     4201 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     4261 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4321 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     4381 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     4441 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4501 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4561 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4621 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4681 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4741 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4801 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4861 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4921 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4981 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5041 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5101 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5161 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5221 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     5281 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5341 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5401 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5461 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5521 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5581 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5641 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5701 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     5761 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5821 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5881 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5941 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6001 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6061 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6121 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6181 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6241 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     6301 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     6361 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     6421 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     6481 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     6541 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     6601 cgaaattaac cctcactaaa gggaacaaaa gctggtaccg ggccccccct cgagatttaa
     6661 atcgccaaaa cgaggcgaag tcgccagggt gcgcgggcac gcagggtaag agccgcaagg
     6721 cctggttcgg taagtattaa gctgtggacg aaatagcagt agccatggcg atgtaattta
     6781 ttagacggcg gcttgataaa agaggactga taatcagtgt attcacagag tcgacggtat
     6841 cgatgtagac gtattaaagc tcaatgatcc tacaagacat taagtaaaaa ggggaagcga
     6901 tcttaaatcc acatgaattt cctttctctt tttcttcttc ttctgttccc tttttctttc
     6961 ttcttccttc aacgtctacg taaatataat gtataacatc tccactttcc tttcccttat
     7021 agcgttatgt tacctgttta aactctaaat tcttcatcta ctactgctgt tactacatgt
     7081 atatatgttc attttttttt ttttttttca ccacggaaaa cgtaaaaatg agagcatttg
     7141 taacaaataa aggagaactg ttcaatcaag tgacatacaa aaactctctc caactctaca
     7201 atggatcccg aataaacaca cataaataaa caaaatggtc gtaattaacg gggtcaaata
     7261 tgcctgtgaa acgtgtatca ggggtcacag ggcggcgcag tgtactcaca ctgatggtcc
     7321 gctacagatg atcagacgca agggaagacc atcgaccaca tgtggccatt gtaaagagct
     7381 gagaagaacc aagaacttca acccatccgg tgggtgcatg tgtgcctctg cacgacggcc
     7441 agctgttggc agcaaggaag atgaaacacg atgtcgttgt gatgagggtg aaccttgtaa
     7501 atgtcatacc aagaggaaaa gcagccggaa atcaaaggga gggtcatgcc acagaagggc
     7561 aaatgatgaa gcagcgcatg tcaatggtct cggtattgca gatctggacg ttcttttggg
     7621 cctaaatggt cgctcgtcgg atgtagacat gacaaccaca ttgccgagtt tgaagccacc
     7681 tctgcaaaac ggagaaatta aggccgacag cattgacaat cttgatttgg cttccctcga
     7741 tccgcttgag caaagcccta gtatatctat ggaacctgtt agtatcaatg aaacaggaag
     7801 cgcatataca actacgaaca cagcactaaa cgatattgac attccattct ccatcaatga
     7861 gttgaacgag ctatacaaac aagtatcttc gcataactca cattca
//
