LOCUS       sequence-348219-        8794 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Vps8-HaloTagx2  in yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8794)
  AUTHORS   Casler JC, Johnson N, Krahn AH, Pantazopoulou A, Day KJ, Glick BS
  TITLE     Clathrin adaptors mediate two sequential pathways of intra-Golgi 
            recycling.
  JOURNAL   J Cell Biol. 2022 Jan 3;221(1). pii: 212747. doi: 
            10.1083/jcb.202103199. Epub 2021 Nov 5.
  PUBMED    34739034
REFERENCE   2  (bases 1 to 8794)
  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: "J Cell 
            Biol."; date: "2022-01-3"; volume: "221(1). pii: 212747. doi"; 
            pages: " 10.1083/jcb.202103199. Epub 2021 Nov 5"
COMMENT     Sequence Label: YIplac211-VPS8-HaloTagx2
FEATURES             Location/Qualifiers
     source          1..8794
                     /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(1480..1485,1486..1503,1504..1510)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1518..1534
                     /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     1523..1545
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1542..1558
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1542..1558
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2454..3344
                     /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             3372..4262
                     /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(5292..5309)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5292..5308)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5301..5323)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5517..5536)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5636..5658
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5776..6579)
                     /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(6580..6795)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6802..6820)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6888..6992
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6993..7061,7062..7853)
                     /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(7211..7230)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8024..8612
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8513..8532
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8766..8783
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattac tgatgaatcc cctaatgatt tttatcaaaa tcattaagtt aaggtagata
      181 cacatcttgt catatgatca aatggtttcg ccaaaaatca ataatcagac aacaaaatgt
      241 gcgaactcga tattttacac gactctcttt accaattctg ccccgaatta cacttaaaac
      301 gactcaacag cttaacgttg gcttgccacg ccttacttga ctgtaaaact ctcactctta
      361 ccgaacttgg ccgtaacctg ccaaccaaag cgagaacaaa acataacatc aaacgaatcg
      421 accgattgtt aggtaatcgt cacctccaca aagagcgact cgctgtatac cgttggcatg
      481 ctagctttat ctgttcgggc aatacgatgc ccattgtact tgttgactgg tctgatatcc
      541 gtgagcaaaa acggcttatg gtattgcgag cttcagtcgc actacacggt cgttctgtta
      601 ctctttatga gaaagcgttc ccgctttcag agcaatgttc aaagaaagct catgaccaat
      661 ttctagccga ccttgcgagc attctaccga gtaacaccac accgctcatt gtcagtgatg
      721 ctggctttaa agtgccatgg tataaatccg ttgagaagct gggttggtac tggttaagtc
      781 gagtaagagg aaaagtacaa tatgcagacc taggagcgga aaactggaaa cctatcagca
      841 acttacatga tatgtcatct agtcactcaa agactttagg ctataagagg ctgactaaaa
      901 gcaatccaat ctcatgccaa attctattgt ataaatctcg ctctaaaggc cgaaaaaatc
      961 agcgctcgac acggactcat tgtcaccacc cgtcacctaa aatctactca gcgtcggcaa
     1021 aggagccatg gattctagca actaacttac ctgttgaaat tcgaacaccc aaacaacttg
     1081 ttaatatcta ttcgaagcga atgcagattg aagaaacctt ccgagacttg aaaagtcctg
     1141 cctacggact aggcctacgc catagccgaa cgagcagctc agagcgtttt gatatcatgc
     1201 tgctaatcgc cctgatgctt caactaacat gttggcttgc gggcgttcat gctcagaaac
     1261 aaggttggga caagcacttc caggctaaca cagtcagaaa tcgaaacgta ctctcaacag
     1321 ttcgcttagg catggaagtt ttgcggcatt ctggctacac aataacaagg gaagacttac
     1381 tcgtggctgc aaccctacta gctcaaaatt tattcacaca tggttacgct ttggggaaat
     1441 tatgagggga tctctcagca ctcattaggc accccaggct ttacacttta tgcttccggc
     1501 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     1561 tgattacgcc aagcttgcat gcagttctca gcaacgctga gagcttacaa attctggact
     1621 tattgaatca agactctaat tttgaagctg cagctataat tcacgaacgc ttggaaagtt
     1681 ttaacctagc agtcagggat ttattaagtt ttattgaaca atgtctaaat gaagggaaaa
     1741 caaatatatc tactttattg gaatctttga ggagggcctt tgatgattgt aattctgctg
     1801 gtaccgagaa aaaatcgtgt tggatattat tgattacatt cctgatcact ctatatggga
     1861 aatatccttc acacgatgaa aggaaagatt tatgtaataa actacttcaa gaagcatttt
     1921 tgggattggt taggtccaag agttcctctc agaaggattc aggtggggaa ttctgggaaa
     1981 taatgtcttc tgttcttgag caccaagacg ttattttaat gaaagttcag gatttaaagc
     2041 aactgctact gaatgttttt aatacttata aattggaaag atctctttct gagttgattc
     2101 aaaagattat agaggattct tcgcaagatc ttgttcaaca gtatagaaaa tttctgagtg
     2161 aagggtggtc tatacacacc gacgactgcg aaatctgcgg gaaaaaaata tggggagctg
     2221 gtctggaccc attacttttt ctagcttggg aaaatgtaca gcgccaccaa gatatgatta
     2281 gtgtagatct caaaactccc cttgtcatat tcaaatgtca ccatggcttt caccagactt
     2341 gcctcgaaaa cttggcccag aaacccgatg aatattcttg tttaatttgc cagacggaat
     2401 ctaacccaaa aatagtaacg gatccggcta gcggaggaag ttcggggagt tcgatggcag
     2461 aaatcggtac tggctttcca ttcgaccccc attatgtgga agtcctgggc gagcgcatgc
     2521 actacgtcga tgttggtccg cgcgatggca cccctgtgct gttcctgcac ggtaacccga
     2581 cctcctccta cgtgtggcgc aacatcatcc cgcatgttgc accgacccat cgctgcattg
     2641 ctccagacct gatcggtatg ggcaaatccg acaaaccaga cctgggttat ttcttcgacg
     2701 accacgtccg cttcatggat gccttcatcg aagccctggg tctggaagag gtcgtcctgg
     2761 tcattcacga ctggggctcc gctctgggtt tccactgggc caagcgcaat ccagagcgcg
     2821 tcaaaggtat tgcatttatg gagttcatcc gccctatccc gacctgggac gaatggccag
     2881 aatttgcccg cgagaccttc caggccttcc gcaccaccga cgtcggccgc aagctgatca
     2941 tcgatcagaa cgtttttatc gagggtacgc tgccgatggg tgtcgtccgc ccgctgactg
     3001 aagtcgagat ggaccattac cgcgagccgt tcctgaatcc tgttgaccgc gagccactgt
     3061 ggcgcttccc aaacgagctg ccaatcgccg gtgagccagc gaacatcgtc gcgctggtcg
     3121 aagaatacat ggactggctg caccagtccc ctgtcccgaa gctgctgttc tggggcaccc
     3181 caggcgttct gatcccaccg gccgaagccg ctcgcctggc caaaagcctg cctaactgca
     3241 aggctgtgga catcggcccg ggtctgaatc tgctgcaaga agacaacccg gacctgatcg
     3301 gcagcgagat cgcgcgctgg ctgtcgacgc tcgagatttc cggctctagc ggaggaagtt
     3361 cggggagttc gatggcagaa atcggtactg gctttccatt cgacccccat tatgtggaag
     3421 tcctgggcga gcgcatgcac tacgtcgatg ttggtccgcg cgatggcacc cctgtgctgt
     3481 tcctgcacgg taacccgacc tcctcctacg tgtggcgcaa catcatcccg catgttgcac
     3541 cgacccatcg ctgcattgct ccagacctga tcggtatggg caaatccgac aaaccagacc
     3601 tgggttattt cttcgacgac cacgtccgct tcatggatgc cttcatcgaa gccctgggtc
     3661 tggaagaggt cgtcctggtc attcacgact ggggctccgc tctgggtttc cactgggcca
     3721 agcgcaatcc agagcgcgtc aaaggtattg catttatgga gttcatccgc cctatcccga
     3781 cctgggacga atggccagaa tttgcccgcg agaccttcca ggccttccgc accaccgacg
     3841 tcggccgcaa gctgatcatc gatcagaacg tttttatcga gggtacgctg ccgatgggtg
     3901 tcgtccgccc gctgactgaa gtcgagatgg accattaccg cgagccgttc ctgaatcctg
     3961 ttgaccgcga gccactgtgg cgcttcccaa acgagctgcc aatcgccggt gagccagcga
     4021 acatcgtcgc gctggtcgaa gaatacatgg actggctgca ccagtcccct gtcccgaagc
     4081 tgctgttctg gggcacccca ggcgttctga tcccaccggc cgaagccgct cgcctggcca
     4141 aaagcctgcc taactgcaag gctgtggaca tcggcccggg tctgaatctg ctgcaagaag
     4201 acaacccgga cctgatcggc agcgagatcg cgcgctggct gtcgacgctc gagatttccg
     4261 gctctagata agcggccgcc atttctaaat atttaataca actttggtta cataaaagta
     4321 aaatttatac acctcatttc attatgtaga ttcatatata gaataccaat tatgattgac
     4381 ccaatagcca tcaaaatcag tagttattaa tacttgtctt tctaggagcc atttgcatat
     4441 ttctgatatt tcatgaagcg aaagtacttc acgacaccta gattgcaatc tactcaatgt
     4501 tatccctgga tgaaatatta tttcgttaac gaccatagta actacctgct tccatatgtt
     4561 tggcctaatg gaaccagatc cattcaccca taaacgagaa aatggtttgc ccagtggaac
     4621 tttgacagca gacttccttg ctgtattcaa ttttgtctga gaattggcat atataatcag
     4681 agggggagtt aatgttcgta tttcaaatct ccttgaagta tacgttaaag gtcgaacatt
     4741 tctcaccatt ggaattacat ccatattcaa tagctctccc gaaatcaaat caattaaaac
     4801 ccaagaggat atatcggacg gctcttgatt gataacaata gcgtttccgg cctccaataa
     4861 ttcattaacc ttacatctat actgaaaagc tacaccaaaa tctttataat ttcctctatt
     4921 ttccaaaatg tctggtaaag tatcagtaca ttcaagtttt gagccatgga gataaatttg
     4981 cttttcctta gccatatcca tgatgacgtt atctattgat tcgtttccaa cgttcttcaa
     5041 cgcctctatt tcatttctag tggtcgaagg actttctatt aatatggacc ggatcactgt
     5101 gcgaatataa tcgtcgcttt gactcttcga taagtcctta gtagaagcgg aaatctttct
     5161 agtgtaagtt ttttttaaag aagagatctc tctttgaatc atagaagaca tggccagatc
     5221 gttgccagaa tgtgtaagtg tgtcatcacg taccagagta aatttttttc tattctcttg
     5281 agctcgaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     5341 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     5401 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg
     5461 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca
     5521 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     5581 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5641 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcggg
     5701 taataactga tataattaaa ttgaagctct aatttgtgag tttagtatac atgcatttac
     5761 ttataataca gttttttagt tttgctggcc gcatcttctc aaatatgctt cccagcctgc
     5821 ttttctgtaa cgttcaccct ctaccttagc atcccttccc tttgcaaata gtcctcttcc
     5881 aacaataata atgtcagatc ctgtagagac cacatcatcc acggttctat actgttgacc
     5941 caatgcgtct cccttgtcat ctaaacccac accgggtgtc ataatcaacc aatcgtaacc
     6001 ttcatctctt ccacccatgt ctctttgagc aataaagccg ataacaaaat ctttgtcgct
     6061 cttcgcaatg tcaacagtac ccttagtata ttctccagta gatagggagc ccttgcatga
     6121 caattctgct aacatcaaaa ggcctctagg ttcctttgtt acttcttctg ccgcctgctt
     6181 caaaccgcta acaatacctg ggcccaccac accgtgtgca ttcgtaatgt ctgcccattc
     6241 tgctattctg tatacacccg cagagtactg caatttgact gtattaccaa tgtcagcaaa
     6301 ttttctgtct tcgaagagta aaaaattgta cttggcggat aatgccttta gcggcttaac
     6361 tgtgccctcc atcgaaaaat cagtcaagat atccacatgt gtttttagta aacaaatttt
     6421 gggacctaat gcttcaacta actccagtaa ttccttggtg gtacgaacat ccaatgaagc
     6481 acacaagttt gtttgctttt cgtgcatgat attaaatagc ttggcagcaa caggactagg
     6541 atgagtagca gcacgttcct tatatgtagc tttcgacatg atttatcttc gtttcctgca
     6601 tgtttttgtt ctgtgcagtt gggttaagaa tactgggcaa tttcatgttt cttcaacact
     6661 acatatgcgt atatatacca atctaagtct gtgctccttc cttcgttctt ccttctgttc
     6721 ggagattacc gaatcaaaaa aatttcaagg aaaccgaaat caaaaaaaag aataaaaaaa
     6781 aaatgatgaa ttgaaaagct ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     6841 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     6901 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     6961 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     7021 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     7081 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     7141 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     7201 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     7261 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     7321 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     7381 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     7441 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     7501 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     7561 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     7621 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     7681 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     7741 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     7801 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     7861 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     7921 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     7981 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     8041 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     8101 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     8161 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     8221 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     8281 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     8341 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     8401 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     8461 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     8521 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     8581 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     8641 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     8701 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     8761 agcgcagcga gtcagtgagc gaggaagcgg aaga
//
