LOCUS       sequence-348260-        8342 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses msGFP2-Ste13  in yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8342)
  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 8342)
  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-msGFP2-STE13
FEATURES             Location/Qualifiers
     source          1..8342
                     /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             734..1441
                     /codon_start=1
                     /product="photostable monomeric superfolder GFP (Valbuena 
                     et al., 2020)"
                     /label=msGFP2
                     /translation="MDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITGGSGGS"
     primer_bind     complement(4840..4857)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4840..4856)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4849..4871)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5065..5084)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5184..5206
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5324..6127)
                     /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="MPKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(6128..6343)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6350..6368)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6436..6540
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6541..6609,6610..7401)
                     /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(6759..6778)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7572..8160
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8061..8080
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8314..8331
                     /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 ccaaatatga
      241 ttataattct gtttaaaaat ggcaacggcg tgtaatactg caagtgactt tgtaaatttg
      301 gaaattgctc cttgaataaa gttttttcca gttgtgtagg ccatttacta gtaaatttgc
      361 cactaggaat gtccgtgatt agtatttgag ttctcatttg cttttggttt gccataattc
      421 ccgattttct aacgctaatg tatctttttt tggtttttga ttgctaccat tatatgcttt
      481 tgtagcatca aggtatatag tataagcctc tctccttatt ctatcaaggc gcggctttac
      541 gcatagccac ccggccgatg aaatttgttt cacggtataa caaacaactt caaaggtgat
      601 tgtacctaga gtcttttaag tatgaaatga cagaaattag atacctattt tgttttgttg
      661 ttttcttcca gcctcatgac ctcaataaac ttagttaggg tgttattcgt gtaaaaaata
      721 tagaaagccc ctaatggata gcactgagag cttgttcaca ggtgttgttc ctatcttggt
      781 cgaattagac ggtgacgtaa acggtcataa gttcagtgtt agaggtgaag gtgaaggtga
      841 cgctactaac ggtaaattga cattgaagtt tatttgtact acaggtaaat tgccagttcc
      901 ttggccaact ttggttacta cattgacata cggtgttcaa tgtttttcaa gatacccaga
      961 tcatatgaag caacatgatt tctttaagtc tgctatgcca gaaggttacg ttcaagaaag
     1021 aactatcact tttaaagatg atggtactta caagacaaga gcagaggtta agttcgaagg
     1081 tgacactttg gttaacagaa tcgaattgaa gggtatcgat tttaaagaag atggtaatat
     1141 tttgggtcat aaattggaat acaactacaa ctcacataac gtttacatta cagctgataa
     1201 acaaaagaat ggtattaaag caaacttcaa gatcagacat aatgttgaag atggttctgt
     1261 tcaattagca gatcattacc aacaaaacac tccaattggt gacggtccag ttttgttacc
     1321 agataaccat tacttgtcta cacaatcaaa attgtctaaa gatccaaacg aaaagagaga
     1381 tcatatggtt ttgttagaat ttgttactgc tgctggtatc acaggtggca gtggcggttc
     1441 ttctgcttca actcattcgc ataaaaggaa gaatagtcat ttatttccgc aaaggaaaag
     1501 ttcaaactct tccatggata agccattctt tcctaataac gatagtgtag ctaacacaga
     1561 tcctcaaagt aacgagaatg gacatacaat taacgagatt agaccgacag aagctacaat
     1621 agatgttaca gatgttcctc aaaccccctt tctccaggaa caatactcaa tgcgtcccag
     1681 acgagaaagc ttccagttta atgatattga aaaccagcat cacacgcatt cctttttcag
     1741 tgtaaacaaa tttaatcgtc gatggggaga atggtctctg cccgagaaaa gatcttatgt
     1801 cttagtcttt acattaatag cactatcagt tttagttcta cttgttattc taataccttc
     1861 aaaacttcta cctaccaaaa tcacgcgtcc taagacatct gcaggagatt ctagtttggg
     1921 caaaagatct ttctcaattg aaaacgtttt gaacggagat tttgccatcc ctgaggatac
     1981 tttccacttt attgacccac cacagcgctt attaggccaa gactcagatc cggggctata
     2041 ttttactaca aaagaaattg atggacatac gaattttatt gcaaagcagt tatttgatga
     2101 gacttttgaa gtaaaccttg ggggcaatag gttcctatat gaaggagtag aatttaccgt
     2161 aagtactgtc caaattaatt ataaattgga taaacttata tttggaacta acttagagtc
     2221 tgagtttaga cactcctcga aggggtttta ttggatcaaa gacttaaaca caggtaatat
     2281 tgaaccgata ttacctcccg aaaaatcaga tgataattat gaacttggat tatcaaaact
     2341 ttcctatgcg catttttctc ctgcatacaa ttatatctat tttgtttacg agaacaatct
     2401 gtttctgcaa caagtaaata gcggagttgc caagaaggta acggaagatg gctccaagga
     2461 tatattcaat gccaaaccag actggatata tgaagaagag gtccttgctt ctgatcaagc
     2521 aatatggtgg gctccagacg attcgaaggc tgtatttgca agattcaatg acaccagtgt
     2581 tgacgatatc cgattaaacc gttacacaaa tatgaacgaa gcataccttt ccgacaccaa
     2641 aatcaaatac ccgaagccag ggttccaaaa tccacagttc gacttgtttc tcgttaactt
     2701 acagaacggt ataatttact caattaatac tggtggtcag aaggattcca ttctgtataa
     2761 cggaaaatgg attagtccag atacattcag gttcgaaatc accgatagaa actctaagat
     2821 attagacgta aaagtgtatg atataccatc atctcaaatg cttactgtga ggaacaccaa
     2881 ttcaaacctg ttcaacggat ggatcgaaaa gacgaaggat attttaagca tccccccaaa
     2941 gccagagttg aaaagaatgg attatgggta cattgatatt catgcagaca gtagagggtt
     3001 cagtcatctc ttttattacc caaccgtttt tgccaaagag cctatccagt tgaccaaagg
     3061 gaactgggaa gtgactggaa atggaatagt cggctacgaa tatgaaacag atactatatt
     3121 ttttactgcc aatgagattg gtgtcatgtc acaacacctt tatagtatca gccttacgga
     3181 ttctacaact cagaatactt tccaaagttt acaaaaccca tcagacaaat acgattttta
     3241 tgacttcgaa ttaagttcta gtgcgagata tgctatttct aaaaaactgg ggcctgatac
     3301 acctattaaa gtagctggtc cgcttacaag ggttttgaac gttgccgaaa ttcacgatga
     3361 ttcaatcttg caactgacga aggatgaaaa gttcaaggaa aaaataaaga actacgattt
     3421 acctattaca agctataaaa ccatggtcct agacgatggc gtagaaataa attacattga
     3481 gatcaaacca gccaatttaa atcctaaaaa aaaatacccg atattagtca acatttatgg
     3541 tggtccagga tctcaaacat ttaccaccaa gtcgtctctt gcgtttgaac aagcagttgt
     3601 ttcaggatta gatgttatcg tcctacaaat agagcccaga ggaactgggg gtaaaggttg
     3661 gagttttaga tcatgggccc gcgaaaaatt aggatattgg gaacctagag acattactga
     3721 agtcacaaaa aaattcatac aaaggaatag ccaacatata gatgaaagca aaattgctat
     3781 ttggggttgg tcatacggcg gttttactag cttgaaaacc gtggaattag acaacggcga
     3841 cacatttaaa tacgccatgg ctgtcgcgcc agtaacaaac tggacattgt atgattccgt
     3901 ttacacggaa agatatatga accaaccatc ggaaaaccat gaaggctatt ttgaagtatc
     3961 caccatccaa aatttcaaat cattcgaatc tttaaagcgt cttttcattg tgcacggaac
     4021 tttcgatgat aatgttcaca tacaaaatac ttttagatta gttgatcaac tgaatctatt
     4081 gggattgacg aactatgaca tgcacatttt tccagactct gatcattcca tccggtacca
     4141 caacgctcaa agaattgtat tccaaaaact atactattgg ttaagggatg cctttgctga
     4201 acgctttgac aacactgaag ttttgcattt ataaaacttt atggatgatt gcgatattaa
     4261 cgataacgat agagacaaac acgcgattaa taatataata atatgaagtt tctcaattgc
     4321 aacgcataat tttggaatga catattaggt acaaatgtta gacctaataa atgtaatatt
     4381 taacgacggt tgtacgtaca ggtacattat tttgtgtact atactaataa gagtatttta
     4441 tatagggcgg tgatgatacc catcgagcta ttatcaccac acagttttcg tatctgtgat
     4501 agaacggctc acaaaatcaa tgagaagaaa agtgtaatta taatcttagt gtatgaataa
     4561 atcatgcttt cgttttcctt ttttcgctac caccaggttt actggtggca gtcttcctct
     4621 tcgtaggttt ggctttctgc ttgataagtt tatccttctt caagtctgtg ctactatctt
     4681 gtgtttcttc atcatctgcg ggaactggat tgtcatctgc gtcaacgaca tcttcgaaat
     4741 cgggggtgct agtactggtg ccaacaccac caccaccaat agtactacct gttctgtaaa
     4801 ttgcgactgg atgtgtcata ctgttgtatt tccgaattca ctggccgtcg ttttacaacg
     4861 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     4921 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     4981 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     5041 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     5101 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5161 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5221 accgaaacgc gcgagacgaa agggcgggta ataactgata taattaaatt gaagctctaa
     5281 tttgtgagtt tagtatacat gcatttactt ataatacagt tttttagttt tgctggccgc
     5341 atcttctcaa atatgcttcc cagcctgctt ttctgtaacg ttcaccctct accttagcat
     5401 cccttccctt tgcaaatagt cctcttccaa caataataat gtcagatcct gtagagacca
     5461 catcatccac ggttctatac tgttgaccca atgcgtctcc cttgtcatct aaacccacac
     5521 cgggtgtcat aatcaaccaa tcgtaacctt catctcttcc acccatgtct ctttgagcaa
     5581 taaagccgat aacaaaatct ttgtcgctct tcgcaatgtc aacagtaccc ttagtatatt
     5641 ctccagtaga tagggagccc ttgcatgaca attctgctaa catcaaaagg cctctaggtt
     5701 cctttgttac ttcttctgcc gcctgcttca aaccgctaac aatacctggg cccaccacac
     5761 cgtgtgcatt cgtaatgtct gcccattctg ctattctgta tacacccgca gagtactgca
     5821 atttgactgt attaccaatg tcagcaaatt ttctgtcttc gaagagtaaa aaattgtact
     5881 tggcggataa tgcctttagc ggcttaactg tgccctccat ggaaaaatca gtcaagatat
     5941 ccacatgtgt ttttagtaaa caaattttag gacctaatgc ttcaactaac tccagtaatt
     6001 ccttggtggt acgaacatcc aatgaagcac acaagtttgt ttgcttttcg tgcatgatat
     6061 taaatagctt ggcagcaaca ggactaggat gagtagcagc acgttcctta tatgtagctt
     6121 tcggcatgat ttatcttcgt ttcctgcatg tttttgttct gtgcagttgg gttaagaata
     6181 ctgggcaatt tcatgtttct tcaacactac atatgcgtat atataccaat ctaagtctgt
     6241 gctccttcct tcgttcttcc ttctgttcgg agattaccga atcaaaaaaa tttcaaggaa
     6301 accgaaatca aaaaaaagaa taaaaaaaaa atgatgaatt gaaaagctgg cctcgtgata
     6361 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     6421 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     6481 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     6541 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     6601 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     6661 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     6721 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     6781 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     6841 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     6901 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     6961 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     7021 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     7081 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     7141 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     7201 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     7261 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     7321 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     7381 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     7441 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     7501 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     7561 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     7621 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     7681 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     7741 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     7801 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     7861 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     7921 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     7981 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     8041 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     8101 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     8161 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     8221 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     8281 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     8341 ga
//
