LOCUS       sequence-445656-        6777 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Gene replacement plasmid to label S. cerevisiae Sec7 with msYFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6777)
  TITLE     Yellow and oxidation-resistant derivatives of a monomeric 
            superfolder GFP
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6777)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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            "Unpublished"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-msYFP2
FEATURES             Location/Qualifiers
     source          1..6777
                     /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             994..1701
                     /codon_start=1
                     /product="photostable monomeric superfolder GFP (Valbuena 
                     et al., 2020)"
                     /label=msGFP2
                     /translation="MDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTSLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTITFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITGGSGGS"
     primer_bind     complement(1036..1057)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1644..1665
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3435..3451)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3435..3451)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3448..3470)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3459..3475
                     /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(3483..3489,3490..3507,3508..3513))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3528..3549
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3666..3683)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3837..4425)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3917..3936)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4596..5387,5388..5456))
                     /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     5219..5238
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5457..5561)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(5691..6491)
                     /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(6492..6707)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     6750..6768
                     /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 accatggata gcactgagag cctgttcacc
     1021 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1081 cgcggcgagg gcgagggcga tgccaccaac ggcaagctga ccctgaagtt catctgcacc
     1141 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacct ccctgggcta cggcctgcag
     1201 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1261 gaaggctacg tccaggagcg caccatcacc ttcaaggacg acggcaccta caagacccgc
     1321 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1381 ttcaaggagg acggcaacat cctggggcac aagctggagt acaacttcaa cagccacaac
     1441 gtctatatca ccgccgacaa gcagaagaac ggcatcaagg ccaacttcaa gatccgccac
     1501 aacgtggagg acggcggcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1561 gacggccccg tgctgctgcc cgacaaccac tacctgagct accagtccaa gctgagcaaa
     1621 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1681 actggaggca gtggcggttc ttaaagcggc cgggctttgt tgaatggtcc agcagattaa
     1741 aatatgctta gttgtagaac acagaaaaaa tgagaaggtt gatatacttt tactcatatt
     1801 taccctgatt taatggcggt gatccttttt agattcgttt aattcataaa caaggtacat
     1861 catatagcaa taacaaatat gtaaactatg tagaaagact tatcaagttt agccatcttg
     1921 gtcctcattc atgggcattt tatactaact tcatttttgc cgaaataaga aaggatgtaa
     1981 tcttaaccta aaatatgtaa gcaattaagg gtgcacgtag ttacttgcga acaaatagga
     2041 aaagaaagaa acaagcacgg atactgaatg actgagtgta ttactacacg atttatctca
     2101 acgaatttaa gtgtggagga cgatcggctg cacgtaggag tcaacgtttt tttactgtgt
     2161 tcatcacaaa accattaagc tgagtcatcc atattgcaaa caataaggta gcaccgcttc
     2221 catcaaaaca ctagaacata ccgatgtttt cacggattta ttggaaacaa atagcagatg
     2281 agggcgtttt tacttgtaat gaaggcgaat gagaaggcaa ggcttacagg cttgttgttt
     2341 atgccaagca tcaatactcg ctatggacga gcgccatcag cgataacaac aaagtttgca
     2401 agtgcttgct tggatataaa aggcttgacg cttaagataa atagggccat acttgcttgc
     2461 ttttactatt tttgttcctc ataagcctga ttgactacgc tattataaac ccctacatta
     2521 tcctataatc aacttcactt gtgtaacgtt gaatgagttc aactgagtaa tactcggtcg
     2581 gaaatatacg ctccatctgg caggaaacat aggctgcatt tatatcttgc acaattatgc
     2641 ccttcgtagg tgtatgtttt catgttttcc attatgtcaa tgtagatgta tctttggaca
     2701 ggcaactttt ggatcaataa agcacaattt ttgagcaatt tgagatatcc agtatttgta
     2761 cgggagtccc aagattgttt agcctaaaag gttgaggact tggattattc ttccatcaag
     2821 cgcctccacc cctcgcatcc tatttctttt gaagcatagg ggacgagact gtctgtaatt
     2881 tatggcgaag tatgacaaga aagattcaaa ctttgtttta cgtatgcccc aataaattct
     2941 atggtagcac tactagagac gtattttggt tttgtaataa ttttcaaact gctcgctatg
     3001 atttgttaca agaaacaaac aaaagacacg accatagggt tcatgttggt ggttgcgaag
     3061 ccacatcttt aaaacattac tgcaaattca gcaggagtga gtagcaaaat cgacaaaaac
     3121 tagagtacgc aacattcccg gtcgtaaatt tcttatcgtg gcgccagaac attcccgtcc
     3181 aatgatgaga atgtctctaa aacaaatgca gtcacagaga ttttctgcga cttctcgagt
     3241 gagaatgttg cttattggtg ctagtggctt gagatcttcg tcaagcaaat tggaatgccc
     3301 ccagattctc aaacaagcac ggcagaaact gaaatacggc caaaattatg aacccttttg
     3361 tgggatcttt gtagaaatag gacctcagga tagtatgatg aagaatgttg gcatgcaagc
     3421 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3481 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3541 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3601 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3661 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3721 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3781 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3841 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3901 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3961 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4021 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4081 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4141 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4201 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4261 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4321 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4381 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4441 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4501 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4561 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4621 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4681 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4741 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4801 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4861 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4921 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4981 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5041 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5101 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5161 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5221 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5281 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5341 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5401 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5461 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5521 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5581 ccacctagct ttttctttcc aatttttttt ttttcgtcat tataaaaatc attacgaccg
     5641 agattcccgg gtaataactg atataattaa attgaagctc taatttgtga gtttagctgg
     5701 ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt aacgttcacc ctctacctta
     5761 gcatcccttc cctttgcaaa tagtcctctt ccaacaataa taatgtcaga tcctgtagag
     5821 accacatcat ccacggttct atactgttga cccaatgcgt ctcccttgtc atctaaaccc
     5881 acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc ttccacccat gtctctttga
     5941 gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa tgtcaacagt acccttagta
     6001 tattctccag tagataggga gcccttgcat gacaattctg ctaacatcaa aaggcctcta
     6061 ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc taacaatacc tgggcccacc
     6121 acaccgtgtg cattcgtaat gtctgcccat tctgctattc tgtatacacc cgcagagtac
     6181 tgcaatttga ctgtattacc aatgtcagca aattttctgt cttcgaagag taaaaaattg
     6241 tacttggcgg ataatgcctt tagcggctta actgtgccct ccatggaaaa atcagtcaag
     6301 atatccacat gtgtttttag taaacaaatt ttgggaccta atgcttcaac taactccagt
     6361 aattccttgg tggtacgaac atccaatgaa gcacacaagt ttgtttgctt ttcgtgcatg
     6421 atattaaata gcttggcagc aacaggacta ggatgagtag cagcacgttc cttatatgta
     6481 gctttcgaca tgatttatct tcgtttcctg caggtttttg ttctgtgcag ttgggttaag
     6541 aatactgggc aatttcatgt ttcttcaaca ctacatatgc gtatatatac caatctaagt
     6601 ctgtgctcct tccttcgttc ttccttctgt tcggagatta ccgaatcaaa aaaatttcaa
     6661 ggaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg aattgaaaag ctctaagaaa
     6721 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc
//
