LOCUS       sequence-495006-        7768 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Insert a C-terminal FRBx2-msGFPx3 tag at KEX2 by popin-popout.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7768)
  TITLE     Glick lab plasmids 2025
REFERENCE   2  (bases 1 to 7768)
  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"
COMMENT     Sequence Label: YIplac211-Kex2-FRBx2-msGFPx3
FEATURES             Location/Qualifiers
     source          1..7768
                     /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"
     regulatory      1059..1068
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1077..1355
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     regulatory      1398..1407
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1416..1694
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             1701..1727
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(4266..4283)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4266..4282)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4275..4297)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4491..4510)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4610..4632
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4750..5553)
                     /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(5554..5769)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5776..5794)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5862..5966
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5967..6035,6036..6827)
                     /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(6185..6204)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6998..7586
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7487..7506
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7740..7757
                     /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 atgcacattg gggtgagaac ggcgtaggtg attggaaaat caaggttaag acaacagaaa
      301 atggacacag gattgacttc cacagttgga ggctgaagct ctttggggaa tccattgatt
      361 catctaaaac agaaactttc gtctttggaa acgataaaga ggaggttgaa ccagctgcta
      421 cagaaagtac cgtatcacaa tattctgcca gttcaacttc tatttccatc agcgctactt
      481 ctacatcttc tatctcaatt ggtgtggaaa cgtcggccat tccccaaacg actactgcga
      541 gtaccgatcc tgattctgat ccaaacactc ctaaaaaact ttcctctcct aggcaagcca
      601 tgcattattt tttaacaata tttttgattg gcgccacatt tttggtgtta tacttcatgt
      661 tttttatgaa atcaaggaga aggatcagaa ggtcaagagc ggaaacgtat gaattcgata
      721 tcattgatac agactctgag tacgattcta ctttggacaa tggaacttcc ggaattactg
      781 agcccgaaga ggttgaggac ttcgattttg atttgtccga tgaagaccat cttgcaagtt
      841 tgtcttcatc agaaaacggt gatgctgaac atacaattga tagtgtacta acaaacgaaa
      901 atccatttag tgaccctata aagcaaaagt tcccaaatga cgccaacgca gaatctgctt
      961 ccaataaatt acaggaatta cagcctgatg ttcctccatc ttccggacga tcgacggatc
     1021 caggttcctc tgggtcttcc ggttcctcag ggtcgtcggc caccatggct tctagaatcc
     1081 tctggcatga gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac tttggggaaa
     1141 ggaacgtgaa aggcatgttt gaggtgctgg agcccttgca tgctatgatg gaacggggcc
     1201 cccagactct gaaggaaaca tcctttaatc aggcctatgg tcgagattta atggaggccc
     1261 aagagtggtg caggaagtac atgaaatcag ggaatgtcaa ggacctcacc caagcctggg
     1321 acctctatta tcatgtgttc cgacgaatct caaagactag tggttcctct gggtcttccg
     1381 gttcctcagg gtcgtcggcc accatggctt ctagaatcct ctggcatgag atgtggcatg
     1441 aaggcctgga agaggcatct cgtttgtact ttggggaaag gaacgtgaaa ggcatgtttg
     1501 aggtgctgga gcccttgcat gctatgatgg aacggggccc ccagactctg aaggaaacat
     1561 cctttaatca ggcctatggt cgagatttaa tggaggccca agagtggtgc aggaagtaca
     1621 tgaaatcagg gaatgtcaag gacctcaccc aagcctggga cctctattat catgtgttcc
     1681 gacgaatctc aaagactagt tatccgtacg acgtaccaga ctacgcagat ccaggaagtt
     1741 cggggtccag cggttcttca ggcgctagca tggatagcac tgagagcctg ttcaccgggg
     1801 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgcgcg
     1861 gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg
     1921 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1981 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2041 gctacgtcca ggagcgcacc atcaccttca aggacgacgg cacctacaag acccgcgccg
     2101 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2161 aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaacagc cacaacgtct
     2221 atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc cgccacaacg
     2281 tggaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2341 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctg agcaaagacc
     2401 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2461 acggcagctc cggctccagc gggtctagca tggatagcac tgagagcctg ttcaccgggg
     2521 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgcgcg
     2581 gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg
     2641 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     2701 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2761 gctacgtcca ggagcgcacc atcaccttca aggacgacgg cacctacaag acccgcgccg
     2821 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2881 aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaacagc cacaacgtct
     2941 atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc cgccacaacg
     3001 tggaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3061 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctg agcaaagacc
     3121 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3181 acggcagctc cggctccagc gggtctagca tggatagcac tgagagcctg ttcaccgggg
     3241 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgcgcg
     3301 gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg
     3361 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     3421 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     3481 gctacgtcca ggagcgcacc atcaccttca aggacgacgg cacctacaag acccgcgccg
     3541 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     3601 aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaacagc cacaacgtct
     3661 atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc cgccacaacg
     3721 tggaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3781 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctg agcaaagacc
     3841 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3901 acggcagctc cggctccagc gggtaaagcg gccgcttcga tatgtacaga aagcttcaaa
     3961 ttacaaaata gcattttttt cttatagatt ataatactct ctcatacgta tacgtatatg
     4021 tgtatatgat atataaacaa acattaatat cctattcctt ccgtttgaaa tccctatgat
     4081 gtactttgca ttgtttgcac ccgcgaataa aatgaaaact ccgaaccgat atatcaagca
     4141 cataaaaggg gagggtccaa ttaatgcata tttaagacca cagctgaata actttaaaac
     4201 ggcagacaaa acaaaaaata ggtcgaataa accttacctg cctagaaggt accgagctcg
     4261 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     4321 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     4381 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt
     4441 ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc
     4501 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     4561 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4621 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cgggtaataa
     4681 ctgatataat taaattgaag ctctaatttg tgagtttagt atacatgcat ttacttataa
     4741 tacagttttt tagttttgct ggccgcatct tctcaaatat gcttcccagc ctgcttttct
     4801 gtaacgttca ccctctacct tagcatccct tccctttgca aatagtcctc ttccaacaat
     4861 aataatgtca gatcctgtag agaccacatc atccacggtt ctatactgtt gacccaatgc
     4921 gtctcccttg tcatctaaac ccacaccggg tgtcataatc aaccaatcgt aaccttcatc
     4981 tcttccaccc atgtctcttt gagcaataaa gccgataaca aaatctttgt cgctcttcgc
     5041 aatgtcaaca gtacccttag tatattctcc agtagatagg gagcccttgc atgacaattc
     5101 tgctaacatc aaaaggcctc taggttcctt tgttacttct tctgccgcct gcttcaaacc
     5161 gctaacaata cctgggccca ccacaccgtg tgcattcgta atgtctgccc attctgctat
     5221 tctgtataca cccgcagagt actgcaattt gactgtatta ccaatgtcag caaattttct
     5281 gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc tttagcggct taactgtgcc
     5341 ctccatggaa aaatcagtca agatatccac atgtgttttt agtaaacaaa ttttgggacc
     5401 taatgcttca actaactcca gtaattcctt ggtggtacga acatccaatg aagcacacaa
     5461 gtttgtttgc ttttcgtgca tgatattaaa tagcttggca gcaacaggac taggatgagt
     5521 agcagcacgt tccttatatg tagctttcga catgatttat cttcgtttcc tgcatgtttt
     5581 tgttctgtgc agttgggtta agaatactgg gcaatttcat gtttcttcaa cactacatat
     5641 gcgtatatat accaatctaa gtctgtgctc cttccttcgt tcttccttct gttcggagat
     5701 taccgaatca aaaaaatttc aaggaaaccg aaatcaaaaa aaagaataaa aaaaaaatga
     5761 tgaattgaaa agctggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     5821 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5881 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5941 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     6001 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     6061 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     6121 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     6181 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     6241 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     6301 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6361 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6421 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6481 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6541 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6601 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6661 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6721 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6781 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6841 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6901 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6961 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     7021 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     7081 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     7141 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     7201 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     7261 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     7321 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     7381 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7441 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7501 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7561 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7621 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     7681 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     7741 gcgagtcagt gagcgaggaa gcggaaga
//
