LOCUS       sequence-311052-        6191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for pop-in/pop-out replacement of Vph1 with Vph1-HaloTag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6191)
  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 6191)
  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-Vph1-HaloTag
FEATURES             Location/Qualifiers
     source          1..6191
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    83..104
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(119..124,125..142,143..149)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    157..173
                     /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     162..184
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     181..197
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     181..197
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             754..1644
                     /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(2666..2683)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2666..2682)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2675..2697)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2891..2910)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3010..3032
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3150..3953)
                     /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(3954..4169)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4176..4194)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4262..4366
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4367..4435,4436..5227)
                     /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(4585..4604)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5398..5986
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5887..5906
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6140..6157
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
       61 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
      121 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
      181 caggaaacag ctatgaccat gattacgcca agcttgcatg cgccatcgac tgaagcagat
      241 gctagttctg aagatttgga agcacaacaa ttaatttccg cgatggacgc cgatgacgct
      301 gaagaagaag aagttggttc tggatctcat ggtgaagact ttggtgatat tatgattcat
      361 caagttattc atacaattga attctgtttg aattgtgttt cgcacactgc atcctattta
      421 cgtttatggg ccttatcatt ggcacatgct caattgtcta gtgttttatg gacaatgaca
      481 attcaaattg cctttggatt tagaggattt gtgggtgtgt ttatgacggt tgcacttttt
      541 gccatgtggt tcgcactaac atgtgcagtt cttgttttga tggaaggtac atctgccatg
      601 cttcattcct tacgtttgca ctgggttgaa tctatgtcca agtttttcgt gggtgaaggt
      661 ttaccatacg aaccattcgc atttgagtat aaagacatgg aagtcgctgt tgctagtgca
      721 agctcttccg cttcaagcgg atccggcggc tccatggcag aaatcggtac tggctttcca
      781 ttcgaccccc attatgtgga agtcctgggc gagcgcatgc actacgtcga tgttggtccg
      841 cgcgatggca cccctgtgct gttcctgcac ggtaacccga cctcctccta cgtgtggcgc
      901 aacatcatcc cgcatgttgc accgacccat cgctgcattg ctccagacct gatcggtatg
      961 ggcaaatccg acaaaccaga cctgggttat ttcttcgacg accacgtccg cttcatggat
     1021 gccttcatcg aagccctggg tctggaagag gtcgtcctgg tcattcacga ctggggctcc
     1081 gctctgggtt tccactgggc caagcgcaat ccagagcgcg tcaaaggtat tgcatttatg
     1141 gagttcatcc gccctatccc gacctgggac gaatggccag aatttgcccg cgagaccttc
     1201 caggccttcc gcaccaccga cgtcggccgc aagctgatca tcgatcagaa cgtttttatc
     1261 gagggtacgc tgccgatggg tgtcgtccgc ccgctgactg aagtcgagat ggaccattac
     1321 cgcgagccgt tcctgaatcc tgttgaccgc gagccactgt ggcgcttccc aaacgagctg
     1381 ccaatcgccg gtgagccagc gaacatcgtc gcgctggtcg aagaatacat ggactggctg
     1441 caccagtccc ctgtcccgaa gctgctgttc tggggcaccc caggcgttct gatcccaccg
     1501 gccgaagccg ctcgcctggc caaaagcctg cctaactgca aggctgtgga catcggcccg
     1561 ggtctgaatc tgctgcaaga agacaacccg gacctgatcg gcagcgagat cgcgcgctgg
     1621 ctgtcgacgc tcgagatttc cggctctaga taagcggccg cattttgagg acttttaaaa
     1681 aaagcgaaac atttaagtac ttcattaaat aataaatgat gctactatat agtgagtaaa
     1741 ccttattata tactatcaat gctaattcaa taaagtattt aaagtatcaa ccaatgctta
     1801 aaaagagtca aaaaaaaggg aaaaagaaat aggaaagcca aacttatctc atgaaaacgt
     1861 tagacttgca atccaatcca ccagtgacca tcatttgcct tggggtaacg ttagaatcga
     1921 aatccacatc caggtctaaa cagttgacaa atcctgtatg caacttttcc caaacgtgta
     1981 agcactgccc cgtgttcaga tcccaacatc tgacagattt atcatcagca caagaaaaca
     2041 agtattggcc tcgaatacta atatctctaa cccacgaaag atgccccttc aacgtaagaa
     2101 cacatctgaa atttgaatct gtaggattcg gtatgggtgc tctatgggcc attaacgtag
     2161 gtagaggtat ctcccagatt tttatcgttc tatctcttga cgctgagaca caatattgca
     2221 tcccccaatt cttatatcga tccgttgatg gtgttctaaa ccttatctcc ggagagtctt
     2281 cgatgaaatg aataaattta actttctcta tgggaaattc atgacccgtg ccaacactaa
     2341 gaccattacc tgatggccaa tgtgttaacc ttaatgtagt gtcatgtgaa ccagagatga
     2401 tgtaatcgcc tagaacgtct atggaacgaa cccattggga atgaggctga aatgttttca
     2461 acgaccaacc attgtggaag tcccaaattt taacagtttg atctctggaa caggacgcta
     2521 tatggacatc attatttttc tgccagattt ttaccgcaga gacaatatgc tcatggccca
     2581 ataaagacct aatttgttgg aacttgcatt cctcggagac ccacttgaaa acatgaatct
     2641 gcaaatcttt agacgcggtg gtaccactgg ccgtcgtttt acaacgtcgt gactgggaaa
     2701 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     2761 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     2821 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     2881 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     2941 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3001 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3061 gacgaaaggg cgggtaataa ctgatataat taaattgaag ctctaatttg tgagtttagt
     3121 atacatgcat ttacttataa tacagttttt tagttttgct ggccgcatct tctcaaatat
     3181 gcttcccagc ctgcttttct gtaacgttca ccctctacct tagcatccct tccctttgca
     3241 aatagtcctc ttccaacaat aataatgtca gatcctgtag agaccacatc atccacggtt
     3301 ctatactgtt gacccaatgc gtctcccttg tcatctaaac ccacaccggg tgtcataatc
     3361 aaccaatcgt aaccttcatc tcttccaccc atgtctcttt gagcaataaa gccgataaca
     3421 aaatctttgt cgctcttcgc aatgtcaaca gtacccttag tatattctcc agtagatagg
     3481 gagcccttgc atgacaattc tgctaacatc aaaaggcctc taggttcctt tgttacttct
     3541 tctgccgcct gcttcaaacc gctaacaata cctgggccca ccacaccgtg tgcattcgta
     3601 atgtctgccc attctgctat tctgtataca cccgcagagt actgcaattt gactgtatta
     3661 ccaatgtcag caaattttct gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc
     3721 tttagcggct taactgtgcc ctccatggaa aaatcagtca agatatccac atgtgttttt
     3781 agtaaacaaa ttttgggacc taatgcttca actaactcca gtaattcctt ggtggtacga
     3841 acatccaatg aagcacacaa gtttgtttgc ttttcgtgca tgatattaaa tagcttggca
     3901 gcaacaggac taggatgagt agcagcacgt tccttatatg tagctttcga catgatttat
     3961 cttcgtttcc tgcatgtttt tgttctgtgc agttgggtta agaatactgg gcaatttcat
     4021 gtttcttcaa cactacatat gcgtatatat accaatctaa gtctgtgctc cttccttcgt
     4081 tcttccttct gttcggagat taccgaatca aaaaaatttc aaggaaaccg aaatcaaaaa
     4141 aaagaataaa aaaaaaatga tgaattgaaa agctggcctc gtgatacgcc tatttttata
     4201 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     4261 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4321 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4381 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4441 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4501 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4561 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4621 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4681 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4741 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4801 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     4861 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     4921 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     4981 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5041 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5101 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5161 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5221 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     5281 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5341 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5401 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5461 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5521 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     5581 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5641 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5701 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5761 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5821 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5881 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5941 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6001 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     6061 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     6121 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     6181 caaaccgcct c
//
