LOCUS       sequence-275841-        7251 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 7251)
  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 7251)
  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..7251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    81..102
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(117..122,123..140,141..147)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    155..171
                     /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     160..182
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     179..195
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     179..195
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1001..1891
                     /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             1919..2809
                     /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(3724..3741)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3724..3740)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3733..3755)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3949..3968)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4068..4090
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4208..5011)
                     /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(5012..5227)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5234..5252)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5320..5424
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5425..5493,5494..6285)
                     /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(5643..5662)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6456..7044
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6945..6964
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7198..7215
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
       61 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
      121 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
      181 ggaaacagct atgaccatga ttacgccaag cttgcatgct ccgttacaac aatcttctgg
      241 tgctttaacg aatgaaatca acttaatcga tttcaatgat ttggacgaag cgccctctca
      301 aggcaacaac aataccaacg gcacgggtac accagcagca gcggaaacat ctgtcaatga
      361 cttattgggc gatttgaccg atttatctat ttcgaaccct tcaaccgcta accaagcatc
      421 ctttggtctg ggaggcgata tcgtgttggg gtcctctcaa cccgcaccac cagttactac
      481 tactaacaac tcgaacaaca ctttagatct tttgggactt tcaactcctc aatcgccaac
      541 taattcccaa gcagtaaaca gtagtgggtt cgatcttttg atgggattta atccaacaac
      601 aggtactact actgcgcccg caagaaccct agtcaatcag tctcctaact tgaagattga
      661 gttcgaaata tctagggagt caaactcagt tataaggata aaatcgttct ttacaaactt
      721 aagttcgtca ccgatctcca acttagtgtt cttattagca gtccctaagt caatgtcttt
      781 gaaattgcaa ccccaatcga gtaactttat gattggcaac gctaaagatg gtatctctca
      841 agaaggtaca attgaaaatg cgccagctaa cccttcaaag gctttgaaag tcaagtggaa
      901 ggtcaactat tctgtcaact ccacccaagc tgaagaaact gctgttttta cgttacctaa
      961 tgtaacggat ccggctagcg gaggaagttc ggggagttcg atggcagaaa tcggtactgg
     1021 ctttccattc gacccccatt atgtggaagt cctgggcgag cgcatgcact acgtcgatgt
     1081 tggtccgcgc gatggcaccc ctgtgctgtt cctgcacggt aacccgacct cctcctacgt
     1141 gtggcgcaac atcatcccgc atgttgcacc gacccatcgc tgcattgctc cagacctgat
     1201 cggtatgggc aaatccgaca aaccagacct gggttatttc ttcgacgacc acgtccgctt
     1261 catggatgcc ttcatcgaag ccctgggtct ggaagaggtc gtcctggtca ttcacgactg
     1321 gggctccgct ctgggtttcc actgggccaa gcgcaatcca gagcgcgtca aaggtattgc
     1381 atttatggag ttcatccgcc ctatcccgac ctgggacgaa tggccagaat ttgcccgcga
     1441 gaccttccag gccttccgca ccaccgacgt cggccgcaag ctgatcatcg atcagaacgt
     1501 ttttatcgag ggtacgctgc cgatgggtgt cgtccgcccg ctgactgaag tcgagatgga
     1561 ccattaccgc gagccgttcc tgaatcctgt tgaccgcgag ccactgtggc gcttcccaaa
     1621 cgagctgcca atcgccggtg agccagcgaa catcgtcgcg ctggtcgaag aatacatgga
     1681 ctggctgcac cagtcccctg tcccgaagct gctgttctgg ggcaccccag gcgttctgat
     1741 cccaccggcc gaagccgctc gcctggccaa aagcctgcct aactgcaagg ctgtggacat
     1801 cggcccgggt ctgaatctgc tgcaagaaga caacccggac ctgatcggca gcgagatcgc
     1861 gcgctggctg tcgacgctcg agatttccgg ctctagcgga ggaagttcgg ggagttcgat
     1921 ggcagaaatc ggtactggct ttccattcga cccccattat gtggaagtcc tgggcgagcg
     1981 catgcactac gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa
     2041 cccgacctcc tcctacgtgt ggcgcaacat catcccgcat gttgcaccga cccatcgctg
     2101 cattgctcca gacctgatcg gtatgggcaa atccgacaaa ccagacctgg gttatttctt
     2161 cgacgaccac gtccgcttca tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt
     2221 cctggtcatt cacgactggg gctccgctct gggtttccac tgggccaagc gcaatccaga
     2281 gcgcgtcaaa ggtattgcat ttatggagtt catccgccct atcccgacct gggacgaatg
     2341 gccagaattt gcccgcgaga ccttccaggc cttccgcacc accgacgtcg gccgcaagct
     2401 gatcatcgat cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg tccgcccgct
     2461 gactgaagtc gagatggacc attaccgcga gccgttcctg aatcctgttg accgcgagcc
     2521 actgtggcgc ttcccaaacg agctgccaat cgccggtgag ccagcgaaca tcgtcgcgct
     2581 ggtcgaagaa tacatggact ggctgcacca gtcccctgtc ccgaagctgc tgttctgggg
     2641 caccccaggc gttctgatcc caccggccga agccgctcgc ctggccaaaa gcctgcctaa
     2701 ctgcaaggct gtggacatcg gcccgggtct gaatctgctg caagaagaca acccggacct
     2761 gatcggcagc gagatcgcgc gctggctgtc gacgctcgag atttccggct ctagataagc
     2821 ggccgctcct ctggcgtttc ttatcaatcc tttctcttct ctatgtatat ttttagaagg
     2881 ataatacctc tatgtagaca atcttataaa gatcttcgca acaatacata tttcacttac
     2941 taatatatgt tgcgttttca tgcatcaaaa gtagttagca atgctagatc gtgtaaagga
     3001 aaaaaaagag aaatagtata attcgccatc ctcataacca ctgaaaaatc gaagttaacc
     3061 aaaaggaaag ttgaaatgga ggaagtgttc cgtttttact cgaactctag aaatatcttc
     3121 attcataaaa gcctttcatt aaaaccatcc accattgatg accccaaaag tggatatggc
     3181 cttttcgtag aaccttcgaa gttcaagaat gatgaattga aaagtgaaac tatccaattg
     3241 ctccgaatcc ctaagcgctg cacgtttaat ataaacacac ttctggctct cttgggagat
     3301 gaggacgaat tttcttcaaa ggaagaattt caaagaacta atgacaaaat taagattgct
     3361 cttcgtgaaa taatggctca tccgaatttc agtgtgttcc tgacagaaac aaatttactc
     3421 ataatttatt tcatgatatt tcaaactatt cgtagtcgtt atgagattcc cgaaaatatc
     3481 caatattatt tagaaaatgt tttaatgagc attgaagtag aaacagccat ggattctatt
     3541 gaaaacttag ctactgatta tggacattat ccacagattt ttggccttcg agagacgcta
     3601 aatttattta aagaactgtt tcacgacgtc ttgaatttaa gtgatatcaa acatctttat
     3661 tctgcaataa tttcacgttg tttagagata ccagaaaggg ccgataccaa aagtgaagaa
     3721 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     3781 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     3841 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct
     3901 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc
     3961 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     4021 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4081 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcg ggtaataact
     4141 gatataatta aattgaagct ctaatttgtg agtttagtat acatgcattt acttataata
     4201 cagtttttta gttttgctgg ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt
     4261 aacgttcacc ctctacctta gcatcccttc cctttgcaaa tagtcctctt ccaacaataa
     4321 taatgtcaga tcctgtagag accacatcat ccacggttct atactgttga cccaatgcgt
     4381 ctcccttgtc atctaaaccc acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc
     4441 ttccacccat gtctctttga gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa
     4501 tgtcaacagt acccttagta tattctccag tagataggga gcccttgcat gacaattctg
     4561 ctaacatcaa aaggcctcta ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc
     4621 taacaatacc tgggcccacc acaccgtgtg cattcgtaat gtctgcccat tctgctattc
     4681 tgtatacacc cgcagagtac tgcaatttga ctgtattacc aatgtcagca aattttctgt
     4741 cttcgaagag taaaaaattg tacttggcgg ataatgcctt tagcggctta actgtgccct
     4801 ccatggaaaa atcagtcaag atatccacat gtgtttttag taaacaaatt ttgggaccta
     4861 atgcttcaac taactccagt aattccttgg tggtacgaac atccaatgaa gcacacaagt
     4921 ttgtttgctt ttcgtgcatg atattaaata gcttggcagc aacaggacta ggatgagtag
     4981 cagcacgttc cttatatgta gctttcgaca tgatttatct tcgtttcctg catgtttttg
     5041 ttctgtgcag ttgggttaag aatactgggc aatttcatgt ttcttcaaca ctacatatgc
     5101 gtatatatac caatctaagt ctgtgctcct tccttcgttc ttccttctgt tcggagatta
     5161 ccgaatcaaa aaaatttcaa ggaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg
     5221 aattgaaaag ctggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     5281 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5341 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5401 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5461 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5521 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5581 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5641 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5701 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5761 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5821 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5881 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5941 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6001 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6061 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6121 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6181 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6241 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6301 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6361 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6421 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6481 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6541 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6601 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6661 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6721 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6781 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6841 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     6901 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     6961 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7021 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7081 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     7141 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     7201 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct c
//
