LOCUS       Exported                7205 bp ds-DNA     circular SYN 07-JAN-2025
DEFINITION  Plasmid for tagging Apl2 with HaloTagx2 by gene replacement.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7205)
  AUTHORS   Glick Lab
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Oct 2, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     Linearize with BstXI for integration at APL2.
FEATURES             Location/Qualifiers
     source          1..7205
                     /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             258..1367
                     /codon_start=1
                     /label=APL2
                     /translation="LVDPSNLKQCTLLLTELKEYAMEYEPEFVSRAIQALSQLGIKYAQ
                     ESFVSKVLDILLELLERQDTIKDDCCISLCDLLRHCPGNDKMAKQVCAVFNTWSNPEVL
                     LQSDIAKCNYVWLLGQHPNNFSDLESKINIFIENFVQEEALTQMSLLMTIVRLHATLTG
                     SMLQSVLELATQQTHELDVRDMAMMYWRCLSMPNNESLVNDLCQNKLPMISNTLEKFSP
                     EVLEKLLMELGTISSIYFKPDSNRRKGKKYVQNIVKGKHIEELESMAKNEISSKANDDV
                     LLDFDERDDVTNTNAGMLNTLTTLGDLDDLFDFGPSEDATQINTNDTKAVQGLKELKLG
                     GDSNGISSGGKNNPDVSGGNIVSQDLLDLF"
     CDS             1404..2294
                     /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             2322..3212
                     /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"
     3'UTR           3230..3683
                     /label=3' APL2
     primer_bind     complement(3698..3714)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(4182..4985)
                     /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(4986..5206)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     promoter        5299..5403
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5404..6264
                     /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      6435..7023
                     /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 ccaagcttgc
      241 atgcctgcag gtcgactcta gtcgatcctt ccaacttgaa acaatgtact ttgttgttga
      301 cagaattaaa ggaatacgca atggaatatg aacctgagtt tgtttcaaga gctattcaag
      361 ctttgtccca gttgggtatt aaatatgcac aagaatcatt cgtcagtaaa gtcttggata
      421 ttctcctaga attgttagaa agacaagaca caataaaaga cgactgctgt atatcattat
      481 gtgatttatt aagacattgc cccggaaacg ataagatggc caaacaagtt tgtgcagtat
      541 ttaatacttg gtcaaatcca gaggtgttat tacaatcaga tattgcgaag tgcaactatg
      601 tctggttgct aggacagcat cccaacaatt tctcagattt ggagtcaaaa atcaatattt
      661 ttatagagaa ctttgttcaa gaggaagctt taacacagat gtctttacta atgacaatag
      721 tcagactaca tgctacttta acgggtagta tgctacaaag cgtcctagaa ttggccacac
      781 aacagacaca tgaattagat gtgcgagata tggctatgat gtattggaga tgcctttcga
      841 tgccaaataa tgaaagtttg gttaatgatc tatgtcaaaa taaacttcca atgatctcca
      901 atacactgga gaagttttca ccagaagtgc tagagaaact attaatggaa ttgggtacta
      961 taagttccat ttatttcaag cccgactcaa ataggagaaa aggcaaaaag tatgttcaaa
     1021 atatagttaa aggaaaacat atagaggaat tagaaagcat ggcaaaaaat gaaatttcta
     1081 gcaaggctaa cgatgatgtg ctattggatt ttgatgaaag agatgatgta acaaatacaa
     1141 atgcaggaat gctgaatact ttaacaactt taggcgattt ggatgattta tttgatttcg
     1201 gaccatctga ggatgccaca caaataaata caaatgatac caaggctgta caagggttga
     1261 aggagctgaa actaggtggc gactcaaacg gtatatcttc tggtggtaaa aacaatcccg
     1321 atgtttcggg cggcaatata gtgtcacagg atctcctcga tttattcacg gatccggcta
     1381 gcggaggaag ttcggggagt tcgatggcag aaatcggtac tggctttcca ttcgaccccc
     1441 attatgtgga agtcctgggc gagcgcatgc actacgtcga tgttggtccg cgcgatggca
     1501 cccctgtgct gttcctgcac ggtaacccga cctcctccta cgtgtggcgc aacatcatcc
     1561 cgcatgttgc accgacccat cgctgcattg ctccagacct gatcggtatg ggcaaatccg
     1621 acaaaccaga cctgggttat ttcttcgacg accacgtccg cttcatggat gccttcatcg
     1681 aagccctggg tctggaagag gtcgtcctgg tcattcacga ctggggctcc gctctgggtt
     1741 tccactgggc caagcgcaat ccagagcgcg tcaaaggtat tgcatttatg gagttcatcc
     1801 gccctatccc gacctgggac gaatggccag aatttgcccg cgagaccttc caggccttcc
     1861 gcaccaccga cgtcggccgc aagctgatca tcgatcagaa cgtttttatc gagggtacgc
     1921 tgccgatggg tgtcgtccgc ccgctgactg aagtcgagat ggaccattac cgcgagccgt
     1981 tcctgaatcc tgttgaccgc gagccactgt ggcgcttccc aaacgagctg ccaatcgccg
     2041 gtgagccagc gaacatcgtc gcgctggtcg aagaatacat ggactggctg caccagtccc
     2101 ctgtcccgaa gctgctgttc tggggcaccc caggcgttct gatcccaccg gccgaagccg
     2161 ctcgcctggc caaaagcctg cctaactgca aggctgtgga catcggcccg ggtctgaatc
     2221 tgctgcaaga agacaacccg gacctgatcg gcagcgagat cgcgcgctgg ctgtcgacgc
     2281 tcgagatttc cggctctagc ggaggaagtt cggggagttc gatggcagaa atcggtactg
     2341 gctttccatt cgacccccat tatgtggaag tcctgggcga gcgcatgcac tacgtcgatg
     2401 ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg taacccgacc tcctcctacg
     2461 tgtggcgcaa catcatcccg catgttgcac cgacccatcg ctgcattgct ccagacctga
     2521 tcggtatggg caaatccgac aaaccagacc tgggttattt cttcgacgac cacgtccgct
     2581 tcatggatgc cttcatcgaa gccctgggtc tggaagaggt cgtcctggtc attcacgact
     2641 ggggctccgc tctgggtttc cactgggcca agcgcaatcc agagcgcgtc aaaggtattg
     2701 catttatgga gttcatccgc cctatcccga cctgggacga atggccagaa tttgcccgcg
     2761 agaccttcca ggccttccgc accaccgacg tcggccgcaa gctgatcatc gatcagaacg
     2821 tttttatcga gggtacgctg ccgatgggtg tcgtccgccc gctgactgaa gtcgagatgg
     2881 accattaccg cgagccgttc ctgaatcctg ttgaccgcga gccactgtgg cgcttcccaa
     2941 acgagctgcc aatcgccggt gagccagcga acatcgtcgc gctggtcgaa gaatacatgg
     3001 actggctgca ccagtcccct gtcccgaagc tgctgttctg gggcacccca ggcgttctga
     3061 tcccaccggc cgaagccgct cgcctggcca aaagcctgcc taactgcaag gctgtggaca
     3121 tcggcccggg tctgaatctg ctgcaagaag acaacccgga cctgatcggc agcgagatcg
     3181 cgcgctggct gtcgacgctc gagatttccg gctctagata agcggccgcc tataaacgtc
     3241 cgttgtagtg aacttaagta ttataccata catatatacc cttcgtatta atttttcctt
     3301 tttattgttg ggctgtgtgt atataattta tgtacaagaa tggatatatc aaaaaatcct
     3361 taaaagatgt aaatcaaaaa catcaatcaa cacagcttat tttgtagagc tttgtttttg
     3421 acataacttt tcaagcttgt tgattttttc taaaacaatt ttatcccttt caccatctaa
     3481 atatttctca tctgtttgat aaaacaggag gcacgatttc atttcatctc gttgcttatc
     3541 acaagtggga aaaactttca ttccatttgc cttagcacaa tctgccattg cttgaacaaa
     3601 atttgcacat tttttgtatg tttccgtctt caagttcttg cgagcctgtt gttcttcgcg
     3661 aggacttagc acccatatag gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc
     3721 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     3781 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     3841 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3901 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     3961 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4021 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4081 cgaaacgcgc gagacgaaag ggcgggtaat aactgatata attaaattga agctctaatt
     4141 tgtgagttta gtatacatgc atttacttat aatacagttt tttagttttg ctggccgcat
     4201 cttctcaaat atgcttccca gcctgctttt ctgtaacgtt caccctctac cttagcatcc
     4261 cttccctttg caaatagtcc tcttccaaca ataataatgt cagatcctgt agagaccaca
     4321 tcatccacgg ttctatactg ttgacccaat gcgtctccct tgtcatctaa acccacaccg
     4381 ggtgtcataa tcaaccaatc gtaaccttca tctcttccac ccatgtctct ttgagcaata
     4441 aagccgataa caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt agtatattct
     4501 ccagtagata gggagccctt gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc
     4561 tttgttactt cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg
     4621 tgtgcattcg taatgtctgc ccattctgct attctgtata cacccgcaga gtactgcaat
     4681 ttgactgtat taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa attgtacttg
     4741 gcggataatg cctttagcgg cttaactgtg ccctccatgg aaaaatcagt caagatatcc
     4801 acatgtgttt ttagtaaaca aattttggga cctaatgctt caactaactc cagtaattcc
     4861 ttggtggtac gaacatccaa tgaagcacac aagtttgttt gcttttcgtg catgatatta
     4921 aatagcttgg cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc
     4981 gacatgattt atcttcgttt cctgcatgtt tttgttctgt gcagttgggt taagaatact
     5041 gggcaatttc atgtttcttc aacactacat atgcgtatat ataccaatct aagtctgtgc
     5101 tccttccttc gttcttcctt ctgttcggag attaccgaat caaaaaaatt tcaaagaaac
     5161 cgaaatcaaa aaaaagaata aaaaaaaaat gatgaattga attgaaaagc tggcctcgtg
     5221 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     5281 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5341 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5401 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5461 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5521 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5581 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     5641 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5701 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5761 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     5821 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     5881 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     5941 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     6001 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     6061 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     6121 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6181 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6241 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     6301 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6361 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6421 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6481 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6541 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     6601 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6661 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6721 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6781 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6841 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     6901 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     6961 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     7021 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     7081 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     7141 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     7201 gaaga
//
