LOCUS       sequence-374608-        7699 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For C-terminal -Halo Tagging of a gene of interest with URA3 
            selection marker in yeast S. cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7699)
  AUTHORS   Ball DA, Mehta GD, Salomon-Kent R, Mazza D, Morisaki T, Mueller F, 
            McNally JG, Karpova TS
  TITLE     Single molecule tracking of Ace1p in Saccharomyces cerevisiae 
            defines a characteristic residence time for non-specific 
            interactions of transcription factors with chromatin.
  JOURNAL   Nucleic Acids Res. 2016 Dec 1;44(21):e160. doi: 10.1093/nar/gkw744. 
            Epub 2016 Aug 26.
  PUBMED    27566148
REFERENCE   2  (bases 1 to 7699)
  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: "Nucleic 
            Acids Res."; date: "2016-12-1"; volume: "44(21):e160. doi"; pages: "
            10.1093/nar/gkw744. Epub 2016 Aug 26"
COMMENT     Sequence Label: pTSK561
FEATURES             Location/Qualifiers
     source          1..7699
                     /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        983..1199
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             1200..2003
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLATGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     primer_bind     complement(2265..2284)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2266..2284)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2291..2308)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2291..2307)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2300..2322)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2448..2903
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2535..2554)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2745..2766
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3163..3825)
                     /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(3826..4013)
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     primer_bind     complement(4160..4179)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4279..4301
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4339..4357)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4425..4529
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4530..4598,4599..5390)
                     /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(4748..4767)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5561..6149
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6050..6069
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6303..6320
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6437..6458
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6473..6478,6479..6496,6497..6503)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6511..6527
                     /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     6516..6538
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6535..6551
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6535..6551
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        6572..6590
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     6618..6634
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     6619..6635
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
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 cgcggggtta atgtggctgt
      961 ggtttcaggg tccataaagc ttttcaattc atcttttttt tttttgttct tttttttgat
     1021 tccggtttct ttgaaatttt tttgattcgg taatctccga gcagaaggaa gaacgaagga
     1081 aggagcacag acttagattg gtatatatac gcatatgtgg tgttgaagaa acgtgaaatt
     1141 gcccagtatt cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca
     1201 tgtcgaaagc tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc
     1261 tatttaatat catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca
     1321 ccaaggaatt actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac
     1381 atgtggatat cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat
     1441 ccgccaagta caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag
     1501 tcaaattgca gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg
     1561 cacacggtgt ggtgggccca ggtattgtta gcggtttgaa gcaggcggcg gaagaagtaa
     1621 caaaggaacc tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctagcta
     1681 ctggagaata tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg
     1741 gctttattgc tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga
     1801 cacccggtgt gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg
     1861 atgatgtggt ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg
     1921 gaagggatgc taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga
     1981 gaagatgcgg ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact
     2041 cacaaattag agcttcaatt taattatatc agttattacc cgggaatctc ggtcgtaatg
     2101 atttctataa tgacgaaaaa aaaaaaattg gaaagaaaaa gcttcatggc ctttataaaa
     2161 aggaactatc caatacctcg ccagaaccaa gtaacagtat tttacggggc acgaatcaag
     2221 aacaataaga caggactgta aagatggacg cgagctccaa ttcgccctat agtgagtcgt
     2281 attacaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     2341 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     2401 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggacg cgccctgtag
     2461 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2521 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2581 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2641 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2701 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2761 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2821 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     2881 caaaatatta acgcttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt
     2941 atttcacacc gcatagatcc gtcgagttca agagaaaaaa aaagaaaaag caaaaagaaa
     3001 aaaggaaagc gcgcctcgtt cagaatgaca cgtatagaat gatgcattac cttgtcatct
     3061 tcagtatcat actgttcgta tacatactta ctgacattca taggtataca tatatacaca
     3121 tgtatatata tcgtatgctg cagctttaaa taatcggtgt cactacataa gaacaccttt
     3181 ggtggaggga acatcgttgg taccattggg cgaggtggct tctcttatgg caaccgcaag
     3241 agccttgaac gcactctcac tacggtgatg atcattcttg cctcgcagac aatcaacgtg
     3301 gagggtaatt ctgctagcct ctgcaaagct ttcaagaaaa tgcgggatca tctcgcaaga
     3361 gagatctcct actttctccc tttgcaaacc aagttcgaca actgcgtacg gcctgttcga
     3421 aagatctacc accgctctgg aaagtgcctc atccaaaggc gcaaatcctg atccaaacct
     3481 ttttactcca cgcacggccc ctagggcctc tttaaaagct tgaccgagag caatcccgca
     3541 gtcttcagtg gtgtgatggt cgtctatgtg taagtcacca atgcactcaa cgattagcga
     3601 ccagccggaa tgcttggcca gagcatgtat catatggtcc agaaacccta tacctgtgtg
     3661 gacgttaatc acttgcgatt gtgtggcctg ttctgctact gcttctgcct ctttttctgg
     3721 gaagatcgag tgctctatcg ctaggggacc accctttaaa gagatcgcaa tctgaatctt
     3781 ggtttcattt gtaatacgct ttactagggc tttctgctct gtcatctttg ccttcgttta
     3841 tcttgcctgc tcatttttta gtatattctt cgaagaaatc acattacttt atataatgta
     3901 taattcatta tgtgataatg ccaatcgcta agaaaaaaaa agagtcatcc gctaggtgga
     3961 aaaaaaaaaa tgaaaatcat taccgaggca taaaaaaata tagagtgtac tagaggaggc
     4021 caagagtaat agaaaaagaa aattgcggga aaggactgtg ttatgacttc cctgactaat
     4081 gccgtgttca aacgatacct ggcagtgact cctagcgctc accaagctct taaaacggga
     4141 atttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     4201 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4261 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4321 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     4381 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     4441 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4501 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     4561 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     4621 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     4681 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     4741 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     4801 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     4861 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     4921 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     4981 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5041 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5101 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5161 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5221 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     5281 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5341 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     5401 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     5461 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5521 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5581 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5641 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5701 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5761 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5821 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5881 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5941 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6001 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6061 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6121 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6181 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     6241 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     6301 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     6361 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     6421 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     6481 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     6541 acagctatga ccatgattac gccaagctcg aaattaaccc tcactaaagg gaacaaaagc
     6601 tggtaccggg ccccccctcg aggtcgacgg tatcgatgta gacgtattaa agctcaatga
     6661 tcctacaaga cattaagtaa aaaggggaag cgatcttaaa tccacatgaa tttcctttct
     6721 ctttttcttc ttcttctgtt ccctttttct ttcttcttcc ttcaacgtct acgtaaatat
     6781 aatgtataac atctccactt tcctttccct tatagcgtta tgttacctgt ttaaactcta
     6841 aattcttcat ctactactgc tgttactaca tgtatatatg ttcatttttt tttttttttt
     6901 tcaccacgga aaacgtaaaa atgagagcat ttgtaacaaa taaaggagaa ctgttcaatc
     6961 aagtgacata caaaaactct ctccaactct acaatggatc ccgaataaac acacataaat
     7021 aaacaaaatg gtcgtaatta acggggtcaa atatgcctgt gaaacgtgta tcaggggtca
     7081 cagggcggcg cagtgtactc acactgatgg tccgctacag atgatcagac gcaagggaag
     7141 accatcgacc acatgtggcc attgtaaaga gctgagaaga accaagaact tcaacccatc
     7201 cggtgggtgc atgtgtgcct ctgcacgacg gccagctgtt ggcagcaagg aagatgaaac
     7261 acgatgtcgt tgtgatgagg gtgaaccttg taaatgtcat accaagagga aaagcagccg
     7321 gaaatcaaag ggagggtcat gccacagaag ggcaaatgat gaagcagcgc atgtcaatgg
     7381 tctcggtatt gcagatctgg acgttctttt gggcctaaat ggtcgctcgt cggatgtaga
     7441 catgacaacc acattgccga gtttgaagcc acctctgcaa aacggagaaa ttaaggccga
     7501 cagcattgac aatcttgatt tggcttccct cgatccgctt gagcaaagcc ctagtatatc
     7561 tatggaacct gttagtatca atgaaacagg aagcgcatat acaactacga acacagcact
     7621 aaacgatatt gacattccat tctccatcaa tgagttgaac gagctataca aacaagtatc
     7681 ttcgcataac tcacattca
//
