LOCUS       sequence-271634-        7256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for pop-in/pop-out replacement of Gga2 with Gga2-HaloTagx2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7256)
  AUTHORS   Casler JC, Glick BS
  TITLE     A microscopy-based kinetic analysis of yeast vacuolar protein 
            sorting.
  JOURNAL   Elife. 2020 Jun 25;9. pii: 56844. doi: 10.7554/eLife.56844.
  PUBMED    32584255
REFERENCE   2  (bases 1 to 7256)
  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: "Elife."; 
            date: "2020-06-25"; volume: "9. pii: 56844. doi"; pages: " 
            10.7554/eLife.56844"
COMMENT     Sequence Label: YIplac211-GGA2-HaloTagx2
FEATURES             Location/Qualifiers
     source          1..7256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1026..1916
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             1944..2834
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     primer_bind     complement(3749..3766)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3749..3765)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3758..3780)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3974..3993)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4093..4115
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4233..5036)
                     /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(5037..5257)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5264..5282)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5350..5454
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5455..5523,5524..6315)
                     /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(5673..5692)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6486..7074
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6975..6994
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7228..7245
                     /label=L4440
                     /note="L4440 vector, forward primer"
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 atgctccgtt acaacaatct tctggtgctt taacgaatga aatcaactta atcgatttca
      301 atgatttgga cgaagcgccc tctcaaggca acaacaatac caacggcacg ggtacaccag
      361 cagcagcgga aacatctgtc aatgacttat tgggcgattt gaccgattta tctatttcga
      421 acccttcaac cgctaaccaa gcatcctttg gtctgggagg cgatatcgtg ttggggtcct
      481 ctcaacccgc accaccagtt actactacta acaactcgaa caacacttta gatcttttgg
      541 gactttcaac tcctcaatcg ccaactaatt cccaagcagt aaacagtagt gggttcgatc
      601 ttttgatggg atttaatcca acaacaggta ctactactgc gcccgcaaga accctagtca
      661 atcagtctcc taacttgaag attgagttcg aaatatctag ggagtcaaac tcagttataa
      721 ggataaaatc gttctttaca aacttaagtt cgtcaccgat ctccaactta gtgttcttat
      781 tagcagtccc taagtcaatg tctttgaaat tgcaacccca atcgagtaac tttatgattg
      841 gcaacgctaa agatggtatc tctcaagaag gtacaattga aaatgcgcca gctaaccctt
      901 caaaggcttt gaaagtcaag tggaaggtca actattctgt caactccacc caagctgaag
      961 aaactgctgt ttttacgtta cctaatgtaa cggatccggc tagcggagga agttcgggga
     1021 gttcgatggc agaaatcggt actggctttc cattcgaccc ccattatgtg gaagtcctgg
     1081 gcgagcgcat gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc
     1141 acggtaaccc gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc
     1201 atcgctgcat tgctccagac ctgatcggta tgggcaaatc cgacaaacca gacctgggtt
     1261 atttcttcga cgaccacgtc cgcttcatgg atgccttcat cgaagccctg ggtctggaag
     1321 aggtcgtcct ggtcattcac gactggggct ccgctctggg tttccactgg gccaagcgca
     1381 atccagagcg cgtcaaaggt attgcattta tggagttcat ccgccctatc ccgacctggg
     1441 acgaatggcc agaatttgcc cgcgagacct tccaggcctt ccgcaccacc gacgtcggcc
     1501 gcaagctgat catcgatcag aacgttttta tcgagggtac gctgccgatg ggtgtcgtcc
     1561 gcccgctgac tgaagtcgag atggaccatt accgcgagcc gttcctgaat cctgttgacc
     1621 gcgagccact gtggcgcttc ccaaacgagc tgccaatcgc cggtgagcca gcgaacatcg
     1681 tcgcgctggt cgaagaatac atggactggc tgcaccagtc ccctgtcccg aagctgctgt
     1741 tctggggcac cccaggcgtt ctgatcccac cggccgaagc cgctcgcctg gccaaaagcc
     1801 tgcctaactg caaggctgtg gacatcggcc cgggtctgaa tctgctgcaa gaagacaacc
     1861 cggacctgat cggcagcgag atcgcgcgct ggctgtcgac gctcgagatt tccggctcta
     1921 gcggaggaag ttcggggagt tcgatggcag aaatcggtac tggctttcca ttcgaccccc
     1981 attatgtgga agtcctgggc gagcgcatgc actacgtcga tgttggtccg cgcgatggca
     2041 cccctgtgct gttcctgcac ggtaacccga cctcctccta cgtgtggcgc aacatcatcc
     2101 cgcatgttgc accgacccat cgctgcattg ctccagacct gatcggtatg ggcaaatccg
     2161 acaaaccaga cctgggttat ttcttcgacg accacgtccg cttcatggat gccttcatcg
     2221 aagccctggg tctggaagag gtcgtcctgg tcattcacga ctggggctcc gctctgggtt
     2281 tccactgggc caagcgcaat ccagagcgcg tcaaaggtat tgcatttatg gagttcatcc
     2341 gccctatccc gacctgggac gaatggccag aatttgcccg cgagaccttc caggccttcc
     2401 gcaccaccga cgtcggccgc aagctgatca tcgatcagaa cgtttttatc gagggtacgc
     2461 tgccgatggg tgtcgtccgc ccgctgactg aagtcgagat ggaccattac cgcgagccgt
     2521 tcctgaatcc tgttgaccgc gagccactgt ggcgcttccc aaacgagctg ccaatcgccg
     2581 gtgagccagc gaacatcgtc gcgctggtcg aagaatacat ggactggctg caccagtccc
     2641 ctgtcccgaa gctgctgttc tggggcaccc caggcgttct gatcccaccg gccgaagccg
     2701 ctcgcctggc caaaagcctg cctaactgca aggctgtgga catcggcccg ggtctgaatc
     2761 tgctgcaaga agacaacccg gacctgatcg gcagcgagat cgcgcgctgg ctgtcgacgc
     2821 tcgagatttc cggctctaga taagcggccg ctcctctggc gtttcttatc aatcctttct
     2881 cttctctatg tatattttta gaaggataat acctctatgt agacaatctt ataaagatct
     2941 tcgcaacaat acatatttca cttactaata tatgttgcgt tttcatgcat caaaagtagt
     3001 tagcaatgct agatcgtgta aaggaaaaaa aagagaaata gtataattcg ccatcctcat
     3061 aaccactgaa aaatcgaagt taaccaaaag gaaagttgaa atggaggaag tgttccgttt
     3121 ttactcgaac tctagaaata tcttcattca taaaagcctt tcattaaaac catccaccat
     3181 tgatgacccc aaaagtggat atggcctttt cgtagaacct tcgaagttca agaatgatga
     3241 attgaaaagt gaaactatcc aattgctccg aatccctaag cgctgcacgt ttaatataaa
     3301 cacacttctg gctctcttgg gagatgagga cgaattttct tcaaaggaag aatttcaaag
     3361 aactaatgac aaaattaaga ttgctcttcg tgaaataatg gctcatccga atttcagtgt
     3421 gttcctgaca gaaacaaatt tactcataat ttatttcatg atatttcaaa ctattcgtag
     3481 tcgttatgag attcccgaaa atatccaata ttatttagaa aatgttttaa tgagcattga
     3541 agtagaaaca gccatggatt ctattgaaaa cttagctact gattatggac attatccaca
     3601 gatttttggc cttcgagaga cgctaaattt atttaaagaa ctgtttcacg acgtcttgaa
     3661 tttaagtgat atcaaacatc tttattctgc aataatttca cgttgtttag agataccaga
     3721 aagggccgat accaaaagtg aagaattcac tggccgtcgt tttacaacgt cgtgactggg
     3781 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     3841 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     3901 aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
     3961 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     4021 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     4081 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     4141 cgagacgaaa gggcgggtaa taactgatat aattaaattg aagctctaat ttgtgagttt
     4201 agtatacatg catttactta taatacagtt ttttagtttt gctggccgca tcttctcaaa
     4261 tatgcttccc agcctgcttt tctgtaacgt tcaccctcta ccttagcatc ccttcccttt
     4321 gcaaatagtc ctcttccaac aataataatg tcagatcctg tagagaccac atcatccacg
     4381 gttctatact gttgacccaa tgcgtctccc ttgtcatcta aacccacacc gggtgtcata
     4441 atcaaccaat cgtaaccttc atctcttcca cccatgtctc tttgagcaat aaagccgata
     4501 acaaaatctt tgtcgctctt cgcaatgtca acagtaccct tagtatattc tccagtagat
     4561 agggagccct tgcatgacaa ttctgctaac atcaaaaggc ctctaggttc ctttgttact
     4621 tcttctgccg cctgcttcaa accgctaaca atacctgggc ccaccacacc gtgtgcattc
     4681 gtaatgtctg cccattctgc tattctgtat acacccgcag agtactgcaa tttgactgta
     4741 ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa aattgtactt ggcggataat
     4801 gcctttagcg gcttaactgt gccctccatg gaaaaatcag tcaagatatc cacatgtgtt
     4861 tttagtaaac aaattttggg acctaatgct tcaactaact ccagtaattc cttggtggta
     4921 cgaacatcca atgaagcaca caagtttgtt tgcttttcgt gcatgatatt aaatagcttg
     4981 gcagcaacag gactaggatg agtagcagca cgttccttat atgtagcttt cgacatgatt
     5041 tatcttcgtt tcctgcatgt ttttgttctg tgcagttggg ttaagaatac tgggcaattt
     5101 catgtttctt caacactaca tatgcgtata tataccaatc taagtctgtg ctccttcctt
     5161 cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat ttcaaagaaa ccgaaatcaa
     5221 aaaaaagaat aaaaaaaaaa tgatgaattg aattgaaaag ctggcctcgt gatacgccta
     5281 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     5341 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5401 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5461 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5521 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5581 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5641 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5701 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5761 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5821 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5881 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5941 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6001 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6061 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6121 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6181 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6241 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6301 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6361 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6421 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6481 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6541 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6601 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6661 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6721 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6781 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6841 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6901 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6961 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7021 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7081 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     7141 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7201 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaaga
//
