LOCUS       sequence-492075-        5572 bp    DNA     circular SYN 06-NOV-2025
DEFINITION  Insert a C-terminal FRBx2 tag at KEX2 by popin-popout.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5572)
  TITLE     Glick lab plasmids 2025
REFERENCE   2  (bases 1 to 5572)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 6, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: YIplac211-Kex2-FRBx2
FEATURES             Location/Qualifiers
     source          1..5572
                     /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(2070..2087)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2070..2086)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2079..2101)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2295..2314)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2414..2436
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2554..3357)
                     /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(3358..3573)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(3580..3598)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3666..3770
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3771..3839,3840..4631)
                     /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(3989..4008)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4802..5390
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5291..5310
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5544..5561
                     /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 ctacgcataa agcggccgct
     1741 tcgatatgta cagaaagctt caaattacaa aatagcattt ttttcttata gattataata
     1801 ctctctcata cgtatacgta tatgtgtata tgatatataa acaaacatta atatcctatt
     1861 ccttccgttt gaaatcccta tgatgtactt tgcattgttt gcacccgcga ataaaatgaa
     1921 aactccgaac cgatatatca agcacataaa aggggagggt ccaattaatg catatttaag
     1981 accacagctg aataacttta aaacggcaga caaaacaaaa aataggtcga ataaacctta
     2041 cctgcctaga aggtaccgag ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg
     2101 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     2161 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     2221 gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     2281 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     2341 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     2401 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     2461 gcgagacgaa agggcgggta ataactgata taattaaatt gaagctctaa tttgtgagtt
     2521 tagtatacat gcatttactt ataatacagt tttttagttt tgctggccgc atcttctcaa
     2581 atatgcttcc cagcctgctt ttctgtaacg ttcaccctct accttagcat cccttccctt
     2641 tgcaaatagt cctcttccaa caataataat gtcagatcct gtagagacca catcatccac
     2701 ggttctatac tgttgaccca atgcgtctcc cttgtcatct aaacccacac cgggtgtcat
     2761 aatcaaccaa tcgtaacctt catctcttcc acccatgtct ctttgagcaa taaagccgat
     2821 aacaaaatct ttgtcgctct tcgcaatgtc aacagtaccc ttagtatatt ctccagtaga
     2881 tagggagccc ttgcatgaca attctgctaa catcaaaagg cctctaggtt cctttgttac
     2941 ttcttctgcc gcctgcttca aaccgctaac aatacctggg cccaccacac cgtgtgcatt
     3001 cgtaatgtct gcccattctg ctattctgta tacacccgca gagtactgca atttgactgt
     3061 attaccaatg tcagcaaatt ttctgtcttc gaagagtaaa aaattgtact tggcggataa
     3121 tgcctttagc ggcttaactg tgccctccat ggaaaaatca gtcaagatat ccacatgtgt
     3181 ttttagtaaa caaattttgg gacctaatgc ttcaactaac tccagtaatt ccttggtggt
     3241 acgaacatcc aatgaagcac acaagtttgt ttgcttttcg tgcatgatat taaatagctt
     3301 ggcagcaaca ggactaggat gagtagcagc acgttcctta tatgtagctt tcgacatgat
     3361 ttatcttcgt ttcctgcatg tttttgttct gtgcagttgg gttaagaata ctgggcaatt
     3421 tcatgtttct tcaacactac atatgcgtat atataccaat ctaagtctgt gctccttcct
     3481 tcgttcttcc ttctgttcgg agattaccga atcaaaaaaa tttcaaggaa accgaaatca
     3541 aaaaaaagaa taaaaaaaaa atgatgaatt gaaaagctgg cctcgtgata cgcctatttt
     3601 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     3661 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3721 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3781 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3841 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3901 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     3961 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4021 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4081 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4141 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4201 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4261 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     4321 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     4381 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4441 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     4501 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     4561 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4621 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4681 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4741 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4801 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4861 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4921 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     4981 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5041 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5101 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5161 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5221 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5281 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5341 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5401 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5461 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5521 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa ga
//
