LOCUS       sequence-445666-        5640 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gene replacement plasmid to label S. cerevisiae Kar2 with moxGFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5640)
  TITLE     Yellow and oxidation-resistant derivatives of a monomeric 
            superfolder GFP
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5640)
  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: 
            "Unpublished"
COMMENT     Sequence Label: YIplac211-Kar2-moxGFP2-HDEL
FEATURES             Location/Qualifiers
     source          1..5640
                     /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             1062..1769
                     /codon_start=1
                     /product="photostable monomeric superfolder GFP (Valbuena 
                     et al., 2020)"
                     /label=msGFP2
                     /translation="MDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FISTTGKLPVPWPTLVTTLTYGVQVFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITGGSGGS"
     primer_bind     complement(1104..1125)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1712..1733
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2138..2155)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2138..2154)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2147..2169)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2363..2382)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2482..2504
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2622..3425)
                     /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(3426..3641)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(3648..3666)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3734..3838
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3839..3907,3908..4699)
                     /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(4057..4076)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4870..5458
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5359..5378
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5612..5629
                     /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 atgcagccgt tcaagctggt gtcttatccg gtgaagaagg tgtcgaagat attgttttat
      301 tggatgtcaa cgctttgact cttggtattg aaaccactgg tggtgtcatg actccattaa
      361 ttaagagaaa tactgctatt cctacaaaga aatcccaaat tttctctact gccgttgaca
      421 accaaccaac cgttatgatc aaggtatacg agggtgaaag agccatgtct aaggacaaca
      481 atctattagg taagtttgaa ttaaccggca ttccaccagc accaagaggt gtacctcaaa
      541 ttgaagtcac atttgcactt gacgctaatg gtattctgaa ggtgtctgcc acagataagg
      601 gaactggtaa atccgaatct atcaccatca ctaacgataa aggtagatta acccaagaag
      661 agattgatag aatggttgaa gaggctgaaa aattcgcttc tgaagacgct tctatcaagg
      721 ccaaggttga atctagaaac aaattagaaa actacgctca ctctttgaaa aaccaagtta
      781 atggtgacct aggtgaaaaa ttggaagaag aagacaagga aaccttatta gatgctgcta
      841 acgatgtttt agaatggtta gatgataact ttgaaaccgc cattgctgaa gactttgatg
      901 aaaagttcga atctttgtcc aaggtcgctt atccaattac ttctaagttg tacggaggtg
      961 ctgatggttc tggtgccgct gattatgacg acgaagatga agatgacgat ggtgattatt
     1021 tcgaaggaag ttcggggtcc agcggttctt caggcagttc catggatagc actgagagcc
     1081 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1141 tcagcgtgcg cggcgagggc gagggcgatg ccaccaacgg caagctgacc ctgaagttca
     1201 tcagcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     1261 gcgtgcaggt gttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     1321 ccatgcccga aggctacgtc caggagcgca ccatcacctt caaggacgac ggcacctaca
     1381 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     1441 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     1501 gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga
     1561 tccgccacaa cgtggaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     1621 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccaagc
     1681 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     1741 ccgggatcac tggaggcagt ggcggttctc acgacgaatt gtagataaaa tagttaaaaa
     1801 tttttgctgc tggaagcttc aaggttgtta atttattgac ttgcatagaa tatctacatt
     1861 tcttctaaaa atacatgcat agctaattca aacttcgagc ttcatacaat tttcgaggag
     1921 attatactga gtatatacgt aaatatatgc attatatgtt ataaaattag aaagatatag
     1981 aaatttcatt gaagagtata gagactgggg ttaaggtact cagtaacagt gtcatcaata
     2041 tgctaatttt gcgtattact tagctctatt gcgcaaatgc aattttttct taccctgata
     2101 atgctttatt tcccgttccg gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc
     2161 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     2221 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     2281 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     2341 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     2401 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     2461 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     2521 cgaaacgcgc gagacgaaag ggcgggtaat aactgatata attaaattga agctctaatt
     2581 tgtgagttta gtatacatgc atttacttat aatacagttt tttagttttg ctggccgcat
     2641 cttctcaaat atgcttccca gcctgctttt ctgtaacgtt caccctctac cttagcatcc
     2701 cttccctttg caaatagtcc tcttccaaca ataataatgt cagatcctgt agagaccaca
     2761 tcatccacgg ttctatactg ttgacccaat gcgtctccct tgtcatctaa acccacaccg
     2821 ggtgtcataa tcaaccaatc gtaaccttca tctcttccac ccatgtctct ttgagcaata
     2881 aagccgataa caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt agtatattct
     2941 ccagtagata gggagccctt gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc
     3001 tttgttactt cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg
     3061 tgtgcattcg taatgtctgc ccattctgct attctgtata cacccgcaga gtactgcaat
     3121 ttgactgtat taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa attgtacttg
     3181 gcggataatg cctttagcgg cttaactgtg ccctccatgg aaaaatcagt caagatatcc
     3241 acatgtgttt ttagtaaaca aattttggga cctaatgctt caactaactc cagtaattcc
     3301 ttggtggtac gaacatccaa tgaagcacac aagtttgttt gcttttcgtg catgatatta
     3361 aatagcttgg cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc
     3421 gacatgattt atcttcgttt cctgcatgtt tttgttctgt gcagttgggt taagaatact
     3481 gggcaatttc atgtttcttc aacactacat atgcgtatat ataccaatct aagtctgtgc
     3541 tccttccttc gttcttcctt ctgttcggag attaccgaat caaaaaaatt tcaaggaaac
     3601 cgaaatcaaa aaaaagaata aaaaaaaaat gatgaattga aaagctggcc tcgtgatacg
     3661 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     3721 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3781 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     3841 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     3901 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     3961 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4021 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     4081 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     4141 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     4201 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     4261 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     4321 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     4381 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     4441 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     4501 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     4561 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     4621 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     4681 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     4741 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4801 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4861 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4921 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4981 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5041 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5101 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5161 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5221 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5281 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5341 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5401 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5461 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     5521 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     5581 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
//
