LOCUS       Exported                5958 bp ds-DNA     circular SYN 18-JAN-2025
DEFINITION  Plasmid for tagging Gnt1 with HaloTag by gene replacement.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5958)
  AUTHORS   Adam Krahn
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Oct 2, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     Linearize with BsrGI for integration at GNT1.
FEATURES             Location/Qualifiers
     source          1..5958
                     /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..850
                     /codon_start=1
                     /label=GNT1
                     /translation="DIISSTSSLSPLFDFQSSKKVGKLKFASNLMVINPSKEAFDEIVN
                     VMLPKILNKKEKYDMDLINEEMYNLKKIIYKQFIFFRKVRKLFKPEVLVLPFARYGLLT
                     GSLRNPRHYSIIYNDVLGYKTLDNDGNDIPVGLNDSVAYSKYIHFSDYPLAKPWNYPSM
                     KEFECIVKEEDAEDSKLEHQACDLWNSVYASYIQSREICLV"
     CDS             860..1750
                     /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             1751..1753
                     /codon_start=1
                     /label=GNT1
                     /translation=""
     3'UTR           1754..2418
                     /label=3' GNT1
     primer_bind     complement(2451..2467)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2935..3738)
                     /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(3739..3954)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     promoter        4052..4156
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4157..5017
                     /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      5188..5776
                     /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 ccaagctgga
      241 cattataagt tctacatcat ctttatcgcc gttatttgat ttccaaagta gtaaaaaggt
      301 tggaaaatta aaatttgctt ctaatttaat ggttattaat ccttcaaagg aggcgtttga
      361 tgaaattgtg aacgttatgc tgcccaagat tttaaacaag aaggaaaagt acgatatgga
      421 tttgatcaat gaagaaatgt acaatttaaa gaagattatt tataaacagt ttattttctt
      481 cagaaaagtc cgaaaacttt tcaaaccaga agtattggtt ttgccatttg caaggtacgg
      541 cttactgacg ggttcattga gaaacccacg gcactattct ataatctata atgacgtttt
      601 gggttataag acattggata atgatggcaa cgatattccg gtaggattaa acgacagtgt
      661 cgcatattct aagtacatac atttttcgga ttatccattg gccaagccct ggaattatcc
      721 atcaatgaag gagtttgaat gtatcgttaa agaagaagac gcggaggatt caaagctaga
      781 gcaccaagct tgtgaccttt ggaactcggt gtacgcttct tacatacagt ccagggaaat
      841 ctgcctcgtt ggaagttcga tggcagaaat cggtacaggt ttcccttttg acccacacta
      901 tgtcgaagta ttaggtgaaa gaatgcacta tgttgatgtc ggtcctagag atggtactcc
      961 agttttgttt ttacatggta acccaacatc ttcatacgtt tggagaaaca tcatcccaca
     1021 tgttgcacca actcatagat gtattgctcc agatttgatt ggtatgggta aatctgataa
     1081 gccagatttg ggttatttct ttgatgatca tgttagattc atggatgctt ttattgaagc
     1141 attgggtttg gaagaagttg ttttggttat tcatgattgg ggttcagcat taggttttca
     1201 ttgggctaag agaaacccag aaagagttaa gggtatcgct tttatggaat tcattagacc
     1261 aattccaact tgggatgaat ggccagaatt cgcaagagaa acattccaag cttttagaac
     1321 tacagatgtt ggtagaaagt tgatcatcga tcaaaacgtt tttattgaag gtactttgcc
     1381 aatgggtgtt gttagaccat taacagaagt tgaaatggat cattacagag aaccattttt
     1441 aaacccagtt gatagagaac cattgtggag atttccaaat gaattaccaa ttgctggtga
     1501 accagcaaac atcgttgctt tggttgaaga atacatggat tggttacatc aatctccagt
     1561 tccaaagttg ttattttggg gtacaccagg tgttttgatt ccaccagcag aagctgcaag
     1621 attggctaag tcattgccaa actgtaaggc tgttgatatt ggtccaggtt taaatttgtt
     1681 acaagaagat aatccagact tgataggtag tgaaatagcc agatggttgt ccacattaga
     1741 aatctcaggt tgattatgag gagaagtgaa aattttcaaa aagtgtaaaa attagtatag
     1801 tcggaaacgc atatatatat atcatagtat tgataatgtt tctgccaaga ttgtgtccgt
     1861 tatcgcattt tctaacgggt aaggtatgta ctaggtgaga gcgggatctt atttcaacat
     1921 gttgtgcttt aatgcttctt tattcaacag cctgtcaacg tcatcaccgt attttgcgtt
     1981 accttttcca ttttctttga aggcataatc aacagtaatt ctgttgttgg cgactaactg
     2041 attgttcaaa gattttattg ccaaatctgc tttttcaaag tcctcaaaat aaacataagc
     2101 acactttaat tttccgtttg ataggtagaa aatttcgggt tcccttatga gcttaccaaa
     2161 tttattgaaa attttcacta attggtcgct atctatcgaa tctgcaaggt tttttatgaa
     2221 caacttagca ataggtagaa tcatgtcctt agaaatattt gaaggtaaat tagttgttcc
     2281 tgtagaattg gttacttgac gaacctttat cagtcggtcg tataatctaa cggtattatt
     2341 cattattttt attgcatatt gagcatctcc ttgattataa aattcgatga atgcgtagcc
     2401 ttggtaagct tgtagaacct agaggatccc cgggtaccga gctcgaattc actggccgtc
     2461 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     2521 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     2581 cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg
     2641 tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag
     2701 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     2761 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     2821 tcaccgtcat caccgaaacg cgcgagacga aagggcgggt aataactgat ataattaaat
     2881 tgaagctcta atttgtgagt ttagtataca tgcatttact tataatacag ttttttagtt
     2941 ttgctggccg catcttctca aatatgcttc ccagcctgct tttctgtaac gttcaccctc
     3001 taccttagca tcccttccct ttgcaaatag tcctcttcca acaataataa tgtcagatcc
     3061 tgtagagacc acatcatcca cggttctata ctgttgaccc aatgcgtctc ccttgtcatc
     3121 taaacccaca ccgggtgtca taatcaacca atcgtaacct tcatctcttc cacccatgtc
     3181 tctttgagca ataaagccga taacaaaatc tttgtcgctc ttcgcaatgt caacagtacc
     3241 cttagtatat tctccagtag atagggagcc cttgcatgac aattctgcta acatcaaaag
     3301 gcctctaggt tcctttgtta cttcttctgc cgcctgcttc aaaccgctaa caatacctgg
     3361 gcccaccaca ccgtgtgcat tcgtaatgtc tgcccattct gctattctgt atacacccgc
     3421 agagtactgc aatttgactg tattaccaat gtcagcaaat tttctgtctt cgaagagtaa
     3481 aaaattgtac ttggcggata atgcctttag cggcttaact gtgccctcca tggaaaaatc
     3541 agtcaagata tccacatgtg tttttagtaa acaaattttg ggacctaatg cttcaactaa
     3601 ctccagtaat tccttggtgg tacgaacatc caatgaagca cacaagtttg tttgcttttc
     3661 gtgcatgata ttaaatagct tggcagcaac aggactagga tgagtagcag cacgttcctt
     3721 atatgtagct ttcgacatga tttatcttcg tttcctgcat gtttttgttc tgtgcagttg
     3781 ggttaagaat actgggcaat ttcatgtttc ttcaacacta catatgcgta tatataccaa
     3841 tctaagtctg tgctccttcc ttcgttcttc cttctgttcg gagattaccg aatcaaaaaa
     3901 atttcaaaga aaccgaaatc aaaaaaaaga ataaaaaaaa aatgatgaat tgaattgaaa
     3961 agctggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     4021 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4081 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4141 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     4201 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     4261 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     4321 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4381 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4441 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     4501 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     4561 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     4621 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     4681 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     4741 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     4801 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     4861 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     4921 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     4981 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5041 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5101 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5161 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5221 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5281 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     5341 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5401 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5461 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5521 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5581 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5641 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5701 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5761 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5821 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5881 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     5941 gagcgaggaa gcggaaga
//
