LOCUS       Exported                6871 bp ds-DNA     circular SYN 07-JAN-2025
DEFINITION  Plasmid for pop-in/pop-out gene replacement of Vrg4 with 
            HaloTag-Vrg4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6871)
  AUTHORS   Glick Lab
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Oct 2, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     Linearize with MluI for pop-in integration at VRG4.
FEATURES             Location/Qualifiers
     source          1..6871
                     /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"
     misc_feature    234..719
                     /label=5' VRG4
     CDS             720..1610
                     /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             1617..2627
                     /codon_start=1
                     /label=VRG4
                     /translation="SELKTGHAGHNPWASVANSGPISILSYCGSSILMTVTNKFVVNLK
                     DFNMNFVMLFVQSLVCTITLIILRILGYAKFRSLNKTDAKNWFPISFLLVLMIYTSSKA
                     LQYLAVPIYTIFKNLTIILIAYGEVLFFGGSVTSMELSSFLLMVLSSVVATWGDQQAVA
                     AKAASLAEGAAGAVASFNPGYFWMFTNCITSALFVLIMRKRIKLTNFKDFDTMFYNNVL
                     ALPILLLFSFCVEDWSSVNLTNNFSNDSLTAMIISGVASVGISYCSGWCVRVTSSTTYS
                     MVGALNKLPIALSGLIFFDAPRNFLSILSIFIGFLSGIIYAVAKQKKQQAQPLRK"
     misc_feature    2628..3358
                     /label=3' VRG4
     primer_bind     complement(3364..3380)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(3848..4651)
                     /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(4652..4872)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     promoter        4965..5069
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5070..5930
                     /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      6101..6689
                     /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 ccaagcttgt
      241 aatggaccat ggcaatgtgc gccatatata tatgcggctc ccttatgagt agaagaaaat
      301 atatgactgg tggtacaaag tggcatggtg gtatatagat gtaggaacac catttgagtt
      361 tgccataatg attaaatttc cctttttata aagaattagc cgcccataac acgaaaaata
      421 tcacagttga cgaaagaaga cacgtcgcct aaacgcgtaa tgaatcgcaa tatacgaaaa
      481 gctaaacggg tttttcagaa tcctcttacc cagccttgga taatagtttt ggccgaaaaa
      541 aaaaaaaaga aagaaaaaaa ttgaaatttc gcgatccgaa caaacaatga acagaaaaac
      601 agaatatata taacgctgcc aagcctattt cttgttgccg attaattgtt gacatcctct
      661 taattaccaa aagagcctaa gaaaacaaac acactaacca cacagtatct ttcgcccgaa
      721 tggctgaaat cggtactggt ttcccttttg accctcatta tgtcgaagtt ttaggtgaaa
      781 gaatgcacta tgtagatgtc ggtccaagag atggtactcc agttttgttt ttacatggta
      841 acccaacatc ttcatacgtt tggagaaaca tcatcccaca tgttgcacca actcatagat
      901 gtattgctcc agatttgatt ggtatgggta aatctgataa gcctgatttg ggttatttct
      961 ttgatgatca tgttagattc atggatgctt ttattgaagc attgggttta gaagaagttg
     1021 ttttggttat tcatgattgg ggttcagcat taggttttca ttgggctaag agaaacccag
     1081 aaagagttaa gggtatcgct tttatggaat tcattagacc aattccaact tgggatgaat
     1141 ggccagaatt cgcaagagaa acattccaag cttttagaac tacagatgtt ggtagaaagt
     1201 tgatcatcga tcaaaacgtt tttattgaag gtactttgcc aatgggtgtt gttagaccat
     1261 taacagaagt tgaaatggat cattacagag aaccattttt aaacccagtt gatagagaac
     1321 cattgtggag atttccaaat gaattaccaa ttgctggtga accagcaaac atcgttgctt
     1381 tggttgaaga atacatggat tggttacatc aatctccagt tccaaagttg ttattttggg
     1441 gtacaccagg tgttttgatt ccaccagcag aagctgcaag attggctaag tcattgccaa
     1501 actgtaaggc tgttgatatt ggtccaggtt taaatttgtt acaagaagat aatccagact
     1561 tgataggttc agaaatcgcc agatggttgt ccacattaga aatctcaggt ggttcttcag
     1621 aattgaaaac aggtcatgca ggccataacc cttgggcttc agttgccaat tccggtccga
     1681 tctctatttt atcctactgt ggttcctcta ttttaatgac ggtgactaac aagttcgtcg
     1741 tcaatttgaa ggatttcaac atgaactttg tcatgctttt cgtgcaatct ttggtttgta
     1801 ctataacctt gattatccta cgtatactgg gctatgcgaa gttccgttca ttaaacaaaa
     1861 cagacgccaa gaactggttc cctatttcct ttttactggt cttgatgatc tacacctctt
     1921 cgaaggcttt acaatacttg gctgttccaa tttacaccat tttcaagaat ttgactatta
     1981 tcttgattgc ttatggtgag gttctctttt ttggtggctc tgtcacctcc atggaattgt
     2041 catcattttt gttgatggtc ctttcttctg tcgttgcaac ttggggtgac cagcaagctg
     2101 tggctgccaa ggctgcttca ttggctgaag gagcagccgg tgctgttgcc tcctttaacc
     2161 caggttattt ctggatgttc accaactgta tcacttctgc attattcgtt cttataatga
     2221 gaaagagaat taagttaact aacttcaagg atttcgacac tatgttttac aacaatgttt
     2281 tggctctacc tattctattg ctgttttctt tctgtgtgga agattggtct tcagttaatt
     2341 tgaccaataa cttttctaac gattcgctaa ctgctatgat catcagtggt gttgcatccg
     2401 tcggtatttc ttactgttcc ggttggtgtg ttcgtgttac ttcgtctact acatattcga
     2461 tggtaggggc tttgaacaag ctgccaattg ccttgtctgg tttgattttc tttgatgctc
     2521 caagaaactt cttatctatt ctctccattt ttattggttt cctatcaggt attatttatg
     2581 ctgttgccaa acaaaagaag caacaagccc aacctttacg taaatgagaa cttacggggg
     2641 gtgcaattta tttttttttt ttggtttatt ttattttata gaggcatcta atgcaagtag
     2701 atttatatac aattatactt aaaattgata taccttagaa caggtcggac acaacgtgtg
     2761 gtaactccaa tatatcacta atgacaatag cgccttctgg tgtttgcccc aaaatttcat
     2821 ttacttcatt ctcaactaaa tgaatacatg ttttcatgcc caatttgata cctgtctcga
     2881 tgtttttccc actgtcatct ataaaataag cattctcgta tcttgccaac ccgctttcct
     2941 tcatggcttt ttcaaatgct ttaacatgtg gtttgcagac cagcgtatct gtcctagagt
     3001 agtcacaata tgtcaatcca tcaaataaat ccgctatacc caacaatctc aggcaacgta
     3061 ttgcgtggtt tttataagcg ttggtgaaaa gccacaattt atcaatcttt cccgattgtc
     3121 tcaatctcaa cagcatattc cttaatggta tatcaggttt caagatatct tgtagtggta
     3181 aggaatcatc cacaagccgg ttatattcca gtgcgttcac tttgtggaac attacaagtc
     3241 cccttatggc aagtccatac tctttgtagt aagagttatt caaaacgtga gcatcttcag
     3301 gtgatagttt caagtgtgtt tgaaagaacc tcagtataga ttgttgcata agatcgtgaa
     3361 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     3421 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     3481 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct
     3541 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc
     3601 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     3661 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3721 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcg ggtaataact
     3781 gatataatta aattgaagct ctaatttgtg agtttagtat acatgcattt acttataata
     3841 cagtttttta gttttgctgg ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt
     3901 aacgttcacc ctctacctta gcatcccttc cctttgcaaa tagtcctctt ccaacaataa
     3961 taatgtcaga tcctgtagag accacatcat ccacggttct atactgttga cccaatgcgt
     4021 ctcccttgtc atctaaaccc acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc
     4081 ttccacccat gtctctttga gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa
     4141 tgtcaacagt acccttagta tattctccag tagataggga gcccttgcat gacaattctg
     4201 ctaacatcaa aaggcctcta ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc
     4261 taacaatacc tgggcccacc acaccgtgtg cattcgtaat gtctgcccat tctgctattc
     4321 tgtatacacc cgcagagtac tgcaatttga ctgtattacc aatgtcagca aattttctgt
     4381 cttcgaagag taaaaaattg tacttggcgg ataatgcctt tagcggctta actgtgccct
     4441 ccatggaaaa atcagtcaag atatccacat gtgtttttag taaacaaatt ttgggaccta
     4501 atgcttcaac taactccagt aattccttgg tggtacgaac atccaatgaa gcacacaagt
     4561 ttgtttgctt ttcgtgcatg atattaaata gcttggcagc aacaggacta ggatgagtag
     4621 cagcacgttc cttatatgta gctttcgaca tgatttatct tcgtttcctg catgtttttg
     4681 ttctgtgcag ttgggttaag aatactgggc aatttcatgt ttcttcaaca ctacatatgc
     4741 gtatatatac caatctaagt ctgtgctcct tccttcgttc ttccttctgt tcggagatta
     4801 ccgaatcaaa aaaatttcaa agaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg
     4861 aattgaattg aaaagctggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     4921 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     4981 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5041 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5101 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5161 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5221 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5281 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5341 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5401 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5461 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5521 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5581 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5641 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5701 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5761 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     5821 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5881 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     5941 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6001 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6061 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6121 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6181 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6241 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6301 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6361 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6421 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6481 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6541 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6601 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6661 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6721 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     6781 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     6841 gcagcgagtc agtgagcgag gaagcggaag a
//
