LOCUS       Exported                8074 bp ds-DNA     circular SYN 06-JAN-2025
DEFINITION  Plasmid for tagging Ric1 with HaloTagx3 by gene replacement.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8074)
  AUTHORS   Adam Krahn
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Oct 2, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     Linearize with PacI for integration at RIC1.
FEATURES             Location/Qualifiers
     source          1..8074
                     /organism="synthetic DNA construct"
                     /lab_host="Saccharomyces cerevisiae"
                     /mol_type="other DNA"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             238..955
                     /codon_start=3
                     /label=RIC1
                     /translation="ARFSKLVVKHQIYLDQLILAKLEDNTDLEDISHNYRFLKPYKFAL
                     EKILSTKILRSDSLDDILKLIKMYDNTDPEHNISPPTHSGMLEIISNCLRKIETKYWNH
                     LFTNLKMTPRDLLALCIEENEAKMLGVLLLVFLNYDEKDLGDDLHFKKSDLGTEESKAL
                     NDNSTKKSEKSVTNLLKDEELMLKVLELLVTSAANATDPIKATDSWDMCFQLIRLLKEL
                     DRENNTQLVQKALERF"
     CDS             963..993
                     /codon_start=1
                     /label=GSS linker
                     /translation="GSSGSSGSSG"
     CDS             999..1889
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /note="yeast codon-optimized"
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             1896..2786
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /note="yeast codon-optimized"
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             2793..3683
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /note="yeast codon-optimized"
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     misc_feature    3702..4557
                     /label=3' RIC1
     primer_bind     complement(4567..4583)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(5051..5854)
                     /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
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(5855..6075)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     promoter        6168..6272
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6273..7133
                     /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"
     rep_origin      7304..7892
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number colE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagctacg
      241 cgcgattttc caaattggtg gtgaaacacc aaatatacct cgaccaatta atcttagcca
      301 aattagaaga caacacagat ttggaggata tttcacacaa ttatcgtttt ttaaagcctt
      361 acaaatttgc actcgaaaag attttgtcta ctaaaatact gcgaagcgac tctttggatg
      421 atatcctgaa attaattaag atgtatgaca acaccgatcc tgagcataat atttcaccac
      481 caacccacag tggtatgtta gaaattatta gtaattgttt aaggaagatc gaaactaaat
      541 actggaacca tctattcact aatctaaaga tgacaccaag agatttatta gcactctgta
      601 tcgaagaaaa tgaggctaaa atgcttggcg tacttctgtt agtgttttta aattacgatg
      661 agaaagattt gggagatgat ctacatttta agaaatcaga tttagggact gaggaaagca
      721 aagctctaaa tgataactct acaaaaaagt cagagaaatc tgtcactaat ttattgaagg
      781 atgaagaatt aatgcttaaa gtcttagagc ttttggtgac aagtgctgca aacgccacag
      841 acccaattaa agctaccgat tcgtgggaca tgtgtttcca attaatacgc ttactcaagg
      901 aattagacag agaaaacaac acacaattgg ttcaaaaagc actcgagaga ttcaaggatc
      961 caggaagttc ggggtccagc ggttcttcag gcgctagcat ggcagaaatc ggtacaggtt
     1021 tcccttttga cccacactat gtcgaagtat taggtgaaag aatgcactat gttgatgtcg
     1081 gtcctagaga tggtactcca gttttgtttt tacatggtaa cccaacatct tcatacgttt
     1141 ggagaaacat catcccacat gttgcaccaa ctcatagatg tattgctcca gatttgattg
     1201 gtatgggtaa atctgataag ccagatttgg gttatttctt tgatgatcat gttagattca
     1261 tggatgcttt tattgaagca ttgggtttgg aagaagttgt tttggttatt catgattggg
     1321 gttcagcatt aggttttcat tgggctaaga gaaacccaga aagagttaag ggtatcgctt
     1381 ttatggaatt cattagacca attccaactt gggatgaatg gccagaattc gcaagagaaa
     1441 cattccaagc ttttagaact acagatgttg gtagaaagtt gatcatcgat caaaacgttt
     1501 ttattgaagg tactttgcca atgggtgttg ttagaccatt aacagaagtt gaaatggatc
     1561 attacagaga accattttta aacccagttg atagagaacc attgtggaga tttccaaatg
     1621 aattaccaat tgctggtgaa ccagcaaaca tcgttgcttt ggttgaagaa tacatggatt
     1681 ggttacatca atctccagtt ccaaagttgt tattttgggg tacaccaggt gttttgattc
     1741 caccagcaga agctgcaaga ttggctaagt cattgccaaa ctgtaaggct gttgatattg
     1801 gtccaggttt aaatttgtta caagaagata atccagactt gataggtagt gaaatagcca
     1861 gatggttgtc cacattagaa atctcaggtt ctagcatggc agaaatcggt acaggtttcc
     1921 cttttgaccc acactatgtc gaagtattag gtgaaagaat gcactatgtt gatgtcggtc
     1981 ctagagatgg tactccagtt ttgtttttac atggtaaccc aacatcttca tacgtttgga
     2041 gaaacatcat cccacatgtt gcaccaactc atagatgtat tgctccagat ttgattggta
     2101 tgggtaaatc tgataagcca gatttgggtt atttctttga tgatcatgtt agattcatgg
     2161 atgcttttat tgaagcattg ggtttggaag aagttgtttt ggttattcat gattggggtt
     2221 cagcattagg ttttcattgg gctaagagaa acccagaaag agttaagggt atcgctttta
     2281 tggaattcat tagaccaatt ccaacttggg atgaatggcc agaattcgca agagaaacat
     2341 tccaagcttt tagaactaca gatgttggta gaaagttgat catcgatcaa aacgttttta
     2401 ttgaaggtac tttgccaatg ggtgttgtta gaccattaac agaagttgaa atggatcatt
     2461 acagagaacc atttttaaac ccagttgata gagaaccatt gtggagattt ccaaatgaat
     2521 taccaattgc tggtgaacca gcaaacatcg ttgctttggt tgaagaatac atggattggt
     2581 tacatcaatc tccagttcca aagttgttat tttggggtac accaggtgtt ttgattccac
     2641 cagcagaagc tgcaagattg gctaagtcat tgccaaactg taaggctgtt gatattggtc
     2701 caggtttaaa tttgttacaa gaagataatc cagacttgat aggtagtgaa atagccagat
     2761 ggttgtccac attagaaatc tcaggttcta gcatggcaga aatcggtaca ggtttccctt
     2821 ttgacccaca ctatgtcgaa gtattaggtg aaagaatgca ctatgttgat gtcggtccta
     2881 gagatggtac tccagttttg tttttacatg gtaacccaac atcttcatac gtttggagaa
     2941 acatcatccc acatgttgca ccaactcata gatgtattgc tccagatttg attggtatgg
     3001 gtaaatctga taagccagat ttgggttatt tctttgatga tcatgttaga ttcatggatg
     3061 cttttattga agcattgggt ttggaagaag ttgttttggt tattcatgat tggggttcag
     3121 cattaggttt tcattgggct aagagaaacc cagaaagagt taagggtatc gcttttatgg
     3181 aattcattag accaattcca acttgggatg aatggccaga attcgcaaga gaaacattcc
     3241 aagcttttag aactacagat gttggtagaa agttgatcat cgatcaaaac gtttttattg
     3301 aaggtacttt gccaatgggt gttgttagac cattaacaga agttgaaatg gatcattaca
     3361 gagaaccatt tttaaaccca gttgatagag aaccattgtg gagatttcca aatgaattac
     3421 caattgctgg tgaaccagca aacatcgttg ctttggttga agaatacatg gattggttac
     3481 atcaatctcc agttccaaag ttgttatttt ggggtacacc aggtgttttg attccaccag
     3541 cagaagctgc aagattggct aagtcattgc caaactgtaa ggctgttgat attggtccag
     3601 gtttaaattt gttacaagaa gataatccag acttgatagg tagtgaaata gccagatggt
     3661 tgtccacatt agaaatctca ggttaatcta gaagcggccg catctatatg acaccatgta
     3721 ttctttacca tatagcttca taacattgac caatcaattt tcagaaatgc gtcgcgcgta
     3781 gttttgcccg atatccccat cccttcagga tctttaaaag gtgatgaaag atgcaccaga
     3841 tagaaaagtt gcattaaaat gattagcaga gatatacaaa tattttagta agaatacata
     3901 aagtatcttt gctttcacaa ataggaacaa gcacataaat acagtataat agacatggct
     3961 gatcaagaaa actctccact acatacagtt ggtttcgatg ctagatttcc ccaacaaaac
     4021 caaacaaagc attgttggca atcttatgtg gattatcaca agtgtgttaa tatgaagggc
     4081 gaagattttg ctccgtgcaa ggtcttttgg aagacctata acgccttatg tcccctagac
     4141 tggatcgaaa aatgggatga tcaaagagaa aaaggtattt tcgcaggtga tatcaactca
     4201 gactaaggat cttagtttcg tttttagtaa tgtgcactta gcctttttta tatcattatt
     4261 attaattact agaattcatt tttgcgtagt agacctacac gggatgaagg atatctatat
     4321 gaaaagtaaa gtaacaatag gagataatga aaaagtgata tcatacgcta acgaatgttc
     4381 atgatggagc caaattccta aaaatcaaac aaacagaaaa aaatgagaat gatgtggggt
     4441 actgaagttt tatttttcat atttattttt aattatagta agtagccatt tctatatata
     4501 ttcaaaaatt atttgaccct cgtaacatct gccactgcac tgaaaagaac aaggcaactc
     4561 gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     4621 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     4681 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
     4741 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     4801 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     4861 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4921 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcgggtaata
     4981 actgatataa ttaaattgaa gctctaattt gtgagtttag tatacatgca tttacttata
     5041 atacagtttt ttagttttgc tggccgcatc ttctcaaata tgcttcccag cctgcttttc
     5101 tgtaacgttc accctctacc ttagcatccc ttccctttgc aaatagtcct cttccaacaa
     5161 taataatgtc agatcctgta gagaccacat catccacggt tctatactgt tgacccaatg
     5221 cgtctccctt gtcatctaaa cccacaccgg gtgtcataat caaccaatcg taaccttcat
     5281 ctcttccacc catgtctctt tgagcaataa agccgataac aaaatctttg tcgctcttcg
     5341 caatgtcaac agtaccctta gtatattctc cagtagatag ggagcccttg catgacaatt
     5401 ctgctaacat caaaaggcct ctaggttcct ttgttacttc ttctgccgcc tgcttcaaac
     5461 cgctaacaat acctgggccc accacaccgt gtgcattcgt aatgtctgcc cattctgcta
     5521 ttctgtatac acccgcagag tactgcaatt tgactgtatt accaatgtca gcaaattttc
     5581 tgtcttcgaa gagtaaaaaa ttgtacttgg cggataatgc ctttagcggc ttaactgtgc
     5641 cctccatgga aaaatcagtc aagatatcca catgtgtttt tagtaaacaa attttgggac
     5701 ctaatgcttc aactaactcc agtaattcct tggtggtacg aacatccaat gaagcacaca
     5761 agtttgtttg cttttcgtgc atgatattaa atagcttggc agcaacagga ctaggatgag
     5821 tagcagcacg ttccttatat gtagctttcg acatgattta tcttcgtttc ctgcatgttt
     5881 ttgttctgtg cagttgggtt aagaatactg ggcaatttca tgtttcttca acactacata
     5941 tgcgtatata taccaatcta agtctgtgct ccttccttcg ttcttccttc tgttcggaga
     6001 ttaccgaatc aaaaaaattt caaagaaacc gaaatcaaaa aaaagaataa aaaaaaaatg
     6061 atgaattgaa ttgaaaagct ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     6121 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     6181 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     6241 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     6301 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     6361 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     6421 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     6481 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     6541 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     6601 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     6661 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     6721 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     6781 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6841 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6901 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6961 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     7021 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     7081 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     7141 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     7201 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     7261 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     7321 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     7381 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     7441 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     7501 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     7561 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     7621 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     7681 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     7741 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     7801 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     7861 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7921 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     7981 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     8041 agcgcagcga gtcagtgagc gaggaagcgg aaga
//
