LOCUS       sequence-225310-        7347 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Generate the vps10-104 allele via pop-in pop-out mutagenesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7347)
  AUTHORS   Casler JC, Papanikou E, Barrero JJ, Glick BS
  TITLE     Maturation-driven transport and AP-1-dependent recycling of a 
            secretory cargo in the Golgi.
  JOURNAL   J Cell Biol. 2019 May 6;218(5):1582-1601. doi: 
            10.1083/jcb.201807195. Epub 2019 Mar 11.
  PUBMED    30858194
REFERENCE   2  (bases 1 to 7347)
  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"; doi: 
            "10.1083/jcb.201807195"; journalName: "J Cell Biol"; date: 
            "2019-05-6- 6"; volume: "218"; issue: "5"; pages: "1582-1601"
COMMENT     Sequence Label: YIplac211-vps10-104
FEATURES             Location/Qualifiers
     source          1..7347
                     /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"
     primer_bind     complement(3845..3862)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3845..3861)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3854..3876)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4070..4089)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4189..4211
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4329..5132)
                     /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(5133..5348)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5355..5373)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5441..5545
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5546..5614,5615..6406)
                     /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(5764..5783)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6577..7165
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7066..7085
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7319..7336
                     /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 atgcctgcag gtcgactcta gaggatcccg tatatatagt ttctgagagg caagagtaaa
      301 aagttatgat atattagtat gttgcttttc tgcaattgtt tcaatgccgt gtaaaaaacc
      361 gcatcatcta aaatctgaga gaatgcagaa acaaaaaaaa aagtaaaaat ttcgagtaaa
      421 agaattttga gttccagggt gaaacagatc taaaactcaa gagcttatgc attgcttgta
      481 ggtacatcac attttacttc agaaagataa aagagtatgt gaaagtaaac tgccctctcc
      541 agttaactcc tcccaaacta aaaagtatcc gcctgtgcgg actacaagca ctaccgcccc
      601 ttgaatgaac accaaaagat gtgtgtaagt taaaaagtcc gaacgaattt atccttatgt
      661 atgttctttt gaacgtcaag tagtcaatct ctccaactac tcgggtcttt atatggatcg
      721 cgtgatcttt taagccttct ttcctctagt ttagtgcagt cgctgctttt ttctcgggct
      781 gagtacggcg aaaagaagga aaaaccaagg tcaaaaaata acctctaaat aacagcatga
      841 tgcaacagct caaaagaacc tctcaagaag caaaaaaaag gagacgagta ggtggtacta
      901 tttcattcta ctaagatctt taaagcaatt cctttccggt ttttttctca actaccgtac
      961 tctttttcct tttttatttt gcccttagat tgtattataa agctctgtat atctggaaaa
     1021 gccctgaagt gtccagtagt catcacacgt tatgatatta cttcattttg tctattctct
     1081 ttgggcctta cttctcattc ctttaactaa tgccgttcca aagaagggaa cgaatgataa
     1141 agaaatagtg gttacagaaa acaaatttga tttcaagatt caattctatc aatactttga
     1201 cacagtcacc gacgaatccc tcctcatgat caattcaaga ggagaagctt atgtatctca
     1261 tgatggtgga caaacaataa aaaggttcga cagtaatggt gaaacaatta ttgaagttgt
     1321 atttaatcca tacttcaatt cttcagctta tctgtttggt tccaaaggta gcattttctc
     1381 tacccatgat aggggatact cttttatgac tgctaaattg cccgaggcta tgcagttagg
     1441 tatgccatta gactttaacg ctaaggcaca ggatacattt atctattatg gtggtaagaa
     1501 ttgtgagtca atcttaagtc cggaatgtca tgcggtagca tacctgacca atgatggggg
     1561 cgaaacgttt acggaaatgc ttgataatgc aattcattgt gagtttgcgg gctcactttt
     1621 caaatatccg tcaaatgagg atatggttat gtgtcaagtg aaggaaaagt cttcgcagac
     1681 aagaagctta gtttcttcta ctgatttttt ccaggatgat aaaaataccg tctttgaaaa
     1741 tattatcggc tacttatcca ctggtggcta tatcatcgtt gctgttcctc atgagaacaa
     1801 cgaattgaga gcatacgtaa ctatcgatgg tactgagttt gccgaggcaa aattcccata
     1861 tgatgaagat gttgggaagc aagaggcatt cactatatta gagtctgaga aaggatcgat
     1921 attcttacat ttagcaacaa acttagtacc aggacgcgat tttggcaatc ttttgaaatc
     1981 caactcaaat ggtacttctt ttgtcacgtt ggagcatgcc gttaatagaa acacattcgg
     2041 ctatgttgac tttgaaaaaa ttcaaggtct cgaaggcatt attctcacca acatcgtttc
     2101 aaatagtgac aaggtcgccg agaataaaga agacaaacaa ttgaagacga agatcacctt
     2161 taatgaaggt tcagattgga actttttgaa acctccgaag agggattcag aaggaaaaaa
     2221 gttttcttgc agctccaaat cactggatga gtgttcattg cacttacatg gctatactga
     2281 acgtaaggat attagagata catattcttc cggttctgca ttaggaatga tgttcggcgt
     2341 tggcaacgtt ggtcctaacc ttttaccata taaagaatgt tccaccttct tcaccaccga
     2401 tggtggcgaa acgtgggctg aagttaagaa gactcctcac caatgggaat acggtgacca
     2461 cggtgggatt ttagttttag ttcctgaaaa ctcagaaact gattctattt cttattctac
     2521 cgattttggt aaaacatgga aagattataa attctgcgct gataaggttt tagtaaagga
     2581 tataactact gttcccaggg attctgcttt gagatttttg ctgtttggag aggcagcaga
     2641 tattggaggc agctcattta gaacgtacac gattgatttt agaaacatct tcgaaagaca
     2701 atgtgatttc gacatcactg gtaaggaaag cgcagattat aaatactctc ctctgggttc
     2761 caaaagcaat tgcctatttg gtcaccaaac cgagtttttg cgtaaaaccg atgaaaattg
     2821 ttttattgga aatattccac tttctgaatt ttcaagaaat atcaaaaact gttcttgtac
     2881 aagacaagat ttcgagtgtg attacaactt ttacaaagct aacgatggta cttgtaaatt
     2941 agtcaaagga ctaagcccag caaatgctgc agacgtttgt aaaaaagagc ctgatttaat
     3001 cgaatatttt gaatcgtcag gctacagaaa gatccctcta tcaacctgtg agggtggcct
     3061 gaaattggat gctccctcat caccacatgc ttgcccagga aaagaaaaag aattcaagga
     3121 aaagtactca gtaagtgccg gtccctttgc atttattttc atttcaattc ttttaataat
     3181 tttctttgcc gcatggtttg tatatgacag aggtatcaga agaaatgggg gatttgcaag
     3241 gtttggagaa attaggctag gtgacgatgg tttaatagaa aacaataata ctgacagagt
     3301 tgtcaataac attgtgaaat caggatttta cgttttctca aatatcgggt ctcttttaca
     3361 gcacacaaaa actaatatag cgcatgctat ttccaaaatt aaaggaaggt ttggaaacag
     3421 aacaggtcca agctactcat ccctgatcca tgatcaattt ttggatgaag cagatgacct
     3481 gcttgctggc cacgatgaag acgccaatga cttatccagt ttcatggatc agggtagtaa
     3541 ttttgaaatc gaagaagatg atgttccaac acttgaagaa gagcatacat catatacaga
     3601 tcaacctacg accaccgatg ttccagatac attaccagaa ggaaatgagg aaaacatcga
     3661 caggcctgat tctacagcgc catctaacga aaaccagtag agagattact ttacatagag
     3721 tagataattc catatacttt tcataaacaa aaacgagcag atcctttaga cagaatatca
     3781 agttctttca taagccaaag aaacttggca tgatcagtac tggtgccggc cgcagctcga
     3841 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     3901 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     3961 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc
     4021 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
     4081 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     4141 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4201 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc gggtaataac
     4261 tgatataatt aaattgaagc tctaatttgt gagtttagta tacatgcatt tacttataat
     4321 acagtttttt agttttgctg gccgcatctt ctcaaatatg cttcccagcc tgcttttctg
     4381 taacgttcac cctctacctt agcatccctt ccctttgcaa atagtcctct tccaacaata
     4441 ataatgtcag atcctgtaga gaccacatca tccacggttc tatactgttg acccaatgcg
     4501 tctcccttgt catctaaacc cacaccgggt gtcataatca accaatcgta accttcatct
     4561 cttccaccca tgtctctttg agcaataaag ccgataacaa aatctttgtc gctcttcgca
     4621 atgtcaacag tacccttagt atattctcca gtagataggg agcccttgca tgacaattct
     4681 gctaacatca aaaggcctct aggttccttt gttacttctt ctgccgcctg cttcaaaccg
     4741 ctaacaatac ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt
     4801 ctgtatacac ccgcagagta ctgcaatttg actgtattac caatgtcagc aaattttctg
     4861 tcttcgaaga gtaaaaaatt gtacttggcg gataatgcct ttagcggctt aactgtgccc
     4921 tccatggaaa aatcagtcaa gatatccaca tgtgttttta gtaaacaaat tttgggacct
     4981 aatgcttcaa ctaactccag taattccttg gtggtacgaa catccaatga agcacacaag
     5041 tttgtttgct tttcgtgcat gatattaaat agcttggcag caacaggact aggatgagta
     5101 gcagcacgtt ccttatatgt agctttcgac atgatttatc ttcgtttcct gcatgttttt
     5161 gttctgtgca gttgggttaa gaatactggg caatttcatg tttcttcaac actacatatg
     5221 cgtatatata ccaatctaag tctgtgctcc ttccttcgtt cttccttctg ttcggagatt
     5281 accgaatcaa aaaaatttca aggaaaccga aatcaaaaaa aagaataaaa aaaaaatgat
     5341 gaattgaaaa gctggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     5401 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5461 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5521 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5581 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5641 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5701 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5761 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5821 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5881 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5941 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6001 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6061 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6121 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6181 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6241 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     6301 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6361 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6421 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6481 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6541 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6601 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6661 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6721 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6781 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6841 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6901 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6961 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7021 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7081 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7141 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7201 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     7261 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     7321 cgagtcagtg agcgaggaag cggaaga
//
