LOCUS       sequence-449369-        6786 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gene replacement plasmid to label S. cerevisiae Sec7 with mEYFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6786)
  TITLE     Yellow and oxidation-resistant derivatives of a monomeric 
            superfolder GFP
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6786)
  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            "Unpublished"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: pUSE-URA3-Sec7-mEYFP
FEATURES             Location/Qualifiers
     source          1..6786
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      988..997
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(994..996,997..999,1000..1713)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1039..1060)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1300..1319)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1647..1668
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3444..3460)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3444..3460)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3457..3479)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3468..3484
                     /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)."
     promoter        complement(join(3492..3498,3499..3516,3517..3522))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3537..3558
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3675..3692)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3846..4434)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3926..3945)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4605..5396,5397..5465))
                     /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     5228..5247
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5466..5570)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(5700..6500)
                     /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
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQLN"
     promoter        complement(6501..6716)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     6759..6777
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ctattgttgt caaatgtgtt
      421 ttacagcttt tgatgataga gctgctcaac gaattattcg agaacgaaga ttttgcccac
      481 tgtatccctt acaaggaagc cattagaatt acaagattgt tggagaaatc gtacgaattt
      541 tctcgtgact ttaatgaaga ttatgggtta agaacaagac tagtagaggc tcgtgtagtt
      601 gataaaatac caaacctgtt gaaacaagaa acgagtgctg ctgcagttct ccttgacatt
      661 atgtttcagt tgtatctgaa cgatgatgag aagaaggctg atttaataac tcgtctgatc
      721 accatttgca tccaggtcgt agagggttat gtttctttgg atgatagaac catggaacgt
      781 agtatcaatg cctggcgttc agtgatagtt gaaatattac aaggttacta cgaatttgat
      841 gatgaagact ttagactata ctgtcctgct atgtatgctc tggtgattca aatattagat
      901 aagagcgttc caacggagtt gagacatgct ataaaacaat ttctaagcag agttggtgaa
      961 ttataccttt ctacggatcc actagtcgcc accatggtga gcaagggcga ggagctgttc
     1021 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1081 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     1141 accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcctg
     1201 cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     1261 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     1321 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1381 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     1441 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     1501 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     1561 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gctaccagtc caagctgagc
     1621 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1681 atcactctcg gcatggacga gctgtacaag taaagcggcc gggctttgtt gaatggtcca
     1741 gcagattaaa atatgcttag ttgtagaaca cagaaaaaat gagaaggttg atatactttt
     1801 actcatattt accctgattt aatggcggtg atccttttta gattcgttta attcataaac
     1861 aaggtacatc atatagcaat aacaaatatg taaactatgt agaaagactt atcaagttta
     1921 gccatcttgg tcctcattca tgggcatttt atactaactt catttttgcc gaaataagaa
     1981 aggatgtaat cttaacctaa aatatgtaag caattaaggg tgcacgtagt tacttgcgaa
     2041 caaataggaa aagaaagaaa caagcacgga tactgaatga ctgagtgtat tactacacga
     2101 tttatctcaa cgaatttaag tgtggaggac gatcggctgc acgtaggagt caacgttttt
     2161 ttactgtgtt catcacaaaa ccattaagct gagtcatcca tattgcaaac aataaggtag
     2221 caccgcttcc atcaaaacac tagaacatac cgatgttttc acggatttat tggaaacaaa
     2281 tagcagatga gggcgttttt acttgtaatg aaggcgaatg agaaggcaag gcttacaggc
     2341 ttgttgttta tgccaagcat caatactcgc tatggacgag cgccatcagc gataacaaca
     2401 aagtttgcaa gtgcttgctt ggatataaaa ggcttgacgc ttaagataaa tagggccata
     2461 cttgcttgct tttactattt ttgttcctca taagcctgat tgactacgct attataaacc
     2521 cctacattat cctataatca acttcacttg tgtaacgttg aatgagttca actgagtaat
     2581 actcggtcgg aaatatacgc tccatctggc aggaaacata ggctgcattt atatcttgca
     2641 caattatgcc cttcgtaggt gtatgttttc atgttttcca ttatgtcaat gtagatgtat
     2701 ctttggacag gcaacttttg gatcaataaa gcacaatttt tgagcaattt gagatatcca
     2761 gtatttgtac gggagtccca agattgttta gcctaaaagg ttgaggactt ggattattct
     2821 tccatcaagc gcctccaccc ctcgcatcct atttcttttg aagcataggg gacgagactg
     2881 tctgtaattt atggcgaagt atgacaagaa agattcaaac tttgttttac gtatgcccca
     2941 ataaattcta tggtagcact actagagacg tattttggtt ttgtaataat tttcaaactg
     3001 ctcgctatga tttgttacaa gaaacaaaca aaagacacga ccatagggtt catgttggtg
     3061 gttgcgaagc cacatcttta aaacattact gcaaattcag caggagtgag tagcaaaatc
     3121 gacaaaaact agagtacgca acattcccgg tcgtaaattt cttatcgtgg cgccagaaca
     3181 ttcccgtcca atgatgagaa tgtctctaaa acaaatgcag tcacagagat tttctgcgac
     3241 ttctcgagtg agaatgttgc ttattggtgc tagtggcttg agatcttcgt caagcaaatt
     3301 ggaatgcccc cagattctca aacaagcacg gcagaaactg aaatacggcc aaaattatga
     3361 acccttttgt gggatctttg tagaaatagg acctcaggat agtatgatga agaatgttgg
     3421 catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3481 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3541 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3601 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3661 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3721 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3781 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3841 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3901 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3961 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4021 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4081 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4141 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4201 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4261 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4321 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4381 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4441 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4501 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4561 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4621 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4681 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4741 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4801 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4861 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4921 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4981 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5041 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5101 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5161 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5221 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5281 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5341 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5401 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5461 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5521 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5581 cgaaaagtgc cacctagctt tttctttcca attttttttt tttcgtcatt ataaaaatca
     5641 ttacgaccga gattcccggg taataactga tataattaaa ttgaagctct aatttgtgag
     5701 tttagctggc cgcatcttct caaatatgct tcccagcctg cttttctgta acgttcaccc
     5761 tctaccttag catcccttcc ctttgcaaat agtcctcttc caacaataat aatgtcagat
     5821 cctgtagaga ccacatcatc cacggttcta tactgttgac ccaatgcgtc tcccttgtca
     5881 tctaaaccca caccgggtgt cataatcaac caatcgtaac cttcatctct tccacccatg
     5941 tctctttgag caataaagcc gataacaaaa tctttgtcgc tcttcgcaat gtcaacagta
     6001 cccttagtat attctccagt agatagggag cccttgcatg acaattctgc taacatcaaa
     6061 aggcctctag gttcctttgt tacttcttct gccgcctgct tcaaaccgct aacaatacct
     6121 gggcccacca caccgtgtgc attcgtaatg tctgcccatt ctgctattct gtatacaccc
     6181 gcagagtact gcaatttgac tgtattacca atgtcagcaa attttctgtc ttcgaagagt
     6241 aaaaaattgt acttggcgga taatgccttt agcggcttaa ctgtgccctc catggaaaaa
     6301 tcagtcaaga tatccacatg tgtttttagt aaacaaattt tgggacctaa tgcttcaact
     6361 aactccagta attccttggt ggtacgaaca tccaatgaag cacacaagtt tgtttgcttt
     6421 tcgtgcatga tattaaatag cttggcagca acaggactag gatgagtagc agcacgttcc
     6481 ttatatgtag ctttcgacat gatttatctt cgtttcctgc aggtttttgt tctgtgcagt
     6541 tgggttaaga atactgggca atttcatgtt tcttcaacac tacatatgcg tatatatacc
     6601 aatctaagtc tgtgctcctt ccttcgttct tccttctgtt cggagattac cgaatcaaaa
     6661 aaatttcaag gaaaccgaaa tcaaaaaaaa gaataaaaaa aaaatgatga attgaaaagc
     6721 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     6781 ttcgtc
//
