LOCUS       sequence-326666-        5649 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast integration vector for the gene replacement of S. cerevisiae 
            KAR2 fused to mEGFP with an ER retrieval signal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5649)
  AUTHORS   Valbuena FM, Fizgerald I, Strack RL, Andruska N, Smith L, Glick BS
  TITLE     A photostable monomeric superfolder GFP.
  JOURNAL   Traffic. 2020 May 15. doi: 10.1111/tra.12737.
  PUBMED    32415747
REFERENCE   2  (bases 1 to 5649)
  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: "Traffic. 
            2020 May 15. doi: 10.1111/tra.12737."
COMMENT     Sequence Label: YIplac211-KAR2-mEGFP-HDEL
FEATURES             Location/Qualifiers
     source          1..5649
                     /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             join(1062..1064,1065..1067,1068..1778)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1107..1128)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1368..1387)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1715..1736
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2147..2164)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2147..2163)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2156..2178)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2372..2391)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2491..2513
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2631..3434)
                     /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(3435..3650)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(3657..3675)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3743..3847
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3848..3916,3917..4708)
                     /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(4066..4085)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4879..5467
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5368..5387
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5621..5638
                     /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 catggtgagc aagggcgagg
     1081 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     1141 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     1201 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     1261 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     1321 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     1381 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     1441 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     1501 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     1561 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     1621 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtcca
     1681 agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     1741 ccgccgggat cactctcggc atggacgagc tgtacaagca cgacgaattg tagataaaat
     1801 agttaaaaat ttttgctgct ggaagcttca aggttgttaa tttattgact tgcatagaat
     1861 atctacattt cttctaaaaa tacatgcata gctaattcaa acttcgagct tcatacaatt
     1921 ttcgaggaga ttatactgag tatatacgta aatatatgca ttatatgtta taaaattaga
     1981 aagatataga aatttcattg aagagtatag agactggggt taaggtactc agtaacagtg
     2041 tcatcaatat gctaattttg cgtattactt agctctattg cgcaaatgca attttttctt
     2101 accctgataa tgctttattt cccgttccgg taccgagctc gaattcactg gccgtcgttt
     2161 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     2221 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     2281 tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg
     2341 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     2401 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     2461 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     2521 cgtcatcacc gaaacgcgcg agacgaaagg gcgggtaata actgatataa ttaaattgaa
     2581 gctctaattt gtgagtttag tatacatgca tttacttata atacagtttt ttagttttgc
     2641 tggccgcatc ttctcaaata tgcttcccag cctgcttttc tgtaacgttc accctctacc
     2701 ttagcatccc ttccctttgc aaatagtcct cttccaacaa taataatgtc agatcctgta
     2761 gagaccacat catccacggt tctatactgt tgacccaatg cgtctccctt gtcatctaaa
     2821 cccacaccgg gtgtcataat caaccaatcg taaccttcat ctcttccacc catgtctctt
     2881 tgagcaataa agccgataac aaaatctttg tcgctcttcg caatgtcaac agtaccctta
     2941 gtatattctc cagtagatag ggagcccttg catgacaatt ctgctaacat caaaaggcct
     3001 ctaggttcct ttgttacttc ttctgccgcc tgcttcaaac cgctaacaat acctgggccc
     3061 accacaccgt gtgcattcgt aatgtctgcc cattctgcta ttctgtatac acccgcagag
     3121 tactgcaatt tgactgtatt accaatgtca gcaaattttc tgtcttcgaa gagtaaaaaa
     3181 ttgtacttgg cggataatgc ctttagcggc ttaactgtgc cctccatgga aaaatcagtc
     3241 aagatatcca catgtgtttt tagtaaacaa attttgggac ctaatgcttc aactaactcc
     3301 agtaattcct tggtggtacg aacatccaat gaagcacaca agtttgtttg cttttcgtgc
     3361 atgatattaa atagcttggc agcaacagga ctaggatgag tagcagcacg ttccttatat
     3421 gtagctttcg acatgattta tcttcgtttc ctgcatgttt ttgttctgtg cagttgggtt
     3481 aagaatactg ggcaatttca tgtttcttca acactacata tgcgtatata taccaatcta
     3541 agtctgtgct ccttccttcg ttcttccttc tgttcggaga ttaccgaatc aaaaaaattt
     3601 caaggaaacc gaaatcaaaa aaaagaataa aaaaaaaatg atgaattgaa aagctggcct
     3661 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     3721 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3781 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     3841 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     3901 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     3961 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     4021 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4081 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4141 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4201 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4261 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4321 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4381 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4441 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4501 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4561 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     4621 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     4681 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     4741 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     4801 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4861 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4921 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4981 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5041 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5101 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5161 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5221 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5281 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5341 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5401 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5461 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5521 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     5581 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     5641 agcggaaga
//
