LOCUS       sequence-492065-        6023 bp    DNA     circular SYN 06-NOV-2025
DEFINITION  Insert a C-terminal FRBx2 tag at SYS1 by popin-popout.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6023)
  TITLE     Glick lab plasmids 2025
REFERENCE   2  (bases 1 to 6023)
  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-Sys1-FRBx2
FEATURES             Location/Qualifiers
     source          1..6023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..29)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(183..771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(263..282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(942..1733,1734..1802))
                     /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     1565..1584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1803..1907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1975..1993
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2000..2215
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             2216..3019
                     /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"
     primer_bind     complement(3137..3159)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3259..3278
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3472..3494
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3486..3503
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3487..3503
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      4593..4602
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4611..4889
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     regulatory      4932..4941
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4950..5228
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             5235..5261
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(5804..5820)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5804..5820)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5817..5839)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5828..5844
                     /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(5852..5858,5859..5876,5877..5882))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5897..5918
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
       61 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      121 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      181 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      241 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      301 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      361 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      421 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      481 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      541 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      601 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
      661 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      721 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      781 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      841 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      901 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      961 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1021 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1081 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1141 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1201 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1261 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1321 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1381 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1441 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1501 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1561 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1621 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1681 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1741 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1801 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1861 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1921 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     1981 cgtatcacga ggccagcttt tcaattcatc attttttttt tattcttttt tttgatttcg
     2041 gtttccttga aatttttttg attcggtaat ctccgaacag aaggaagaac gaaggaagga
     2101 gcacagactt agattggtat atatacgcat atgtagtgtt gaagaaacat gaaattgccc
     2161 agtattctta acccaactgc acagaacaaa aacatgcagg aaacgaagat aaatcatgtc
     2221 gaaagctaca tataaggaac gtgctgctac tcatcctagt cctgttgctg ccaagctatt
     2281 taatatcatg cacgaaaagc aaacaaactt gtgtgcttca ttggatgttc gtaccaccaa
     2341 ggaattactg gagttagttg aagcattagg tcccaaaatt tgtttactaa aaacacatgt
     2401 ggatatcttg actgattttt ccatggaggg cacagttaag ccgctaaagg cattatccgc
     2461 caagtacaat tttttactct tcgaagacag aaaatttgct gacattggta atacagtcaa
     2521 attgcagtac tctgcgggtg tatacagaat agcagaatgg gcagacatta cgaatgcaca
     2581 cggtgtggtg ggcccaggta ttgttagcgg tttgaagcag gcggcagaag aagtaacaaa
     2641 ggaacctaga ggccttttga tgttagcaga attgtcatgc aagggctccc tatctactgg
     2701 agaatatact aagggtactg ttgacattgc gaagagcgac aaagattttg ttatcggctt
     2761 tattgctcaa agagacatgg gtggaagaga tgaaggttac gattggttga ttatgacacc
     2821 cggtgtgggt ttagatgaca agggagacgc attgggtcaa cagtatagaa ccgtggatga
     2881 tgtggtctct acaggatctg acattattat tgttggaaga ggactatttg caaagggaag
     2941 ggatgctaag gtagagggtg aacgttacag aaaagcaggc tgggaagcat atttgagaag
     3001 atgcggccag caaaactaaa aaactgtatt ataagtaaat gcatgtatac taaactcaca
     3061 aattagagct tcaatttaat tatatcagtt attacccgcc ctttcgtctc gcgcgtttcg
     3121 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     3181 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     3241 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt
     3301 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca
     3361 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     3421 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
     3481 gacgttgtaa aacgacggcc agtgaattcc tggtaatatt tcagttgttg actaatatat
     3541 atagtggttt ttgccaaaat taaagacttc cttttattaa ctaggtgacc agatataagg
     3601 aagtgtattt gctttctaat aacttgtgta cggccataac ccggctttta atcagtttat
     3661 ttaacaaggt atacaaatcg ccaaacgtaa caagagatag gcgctattat tcatttaata
     3721 ttgtcgtatt caaaatacta acggtgaaca atttgctcca ttgtaacact ataggacaag
     3781 gctaatcatg aataaatatc tccccaccca aaatattcgt agctcgcgat attcacggta
     3841 atattctgta acttcatgtt aagagatgat ctttaggaag gctaaggaag ggtacacata
     3901 gaagtgagat aaaaaatagg catttgaatt actgtaacat ggtttcgata agaaggtatt
     3961 tgcgagtgcc gaatgagttg aaaccttccc agatcttcaa acaagattct ctctctccaa
     4021 gtaagattgg acttcaaatt gttcttttac agatttttta ttacaccaca gcgatcgttc
     4081 ttttttattg ttgggcgaag ctagcgggat atgatctcaa tatcaaggaa tggctgtttt
     4141 cttgggagaa tatcgacttt acgaacgcat acggtttatc aatctcactg ttatggcttc
     4201 tggattcatt aatatgtgtt tttttcttaa ctgttatcgt tggacggagt aaactcgcat
     4261 gggattttgc tatcactata catgccatca attttattgt ggtgttcctc tatactcgga
     4321 aatttccttc tttttcttgg ttttttcttc aaattttatc atctttaata ctaatatttt
     4381 tggggacatg gacaacaaga tggagagagc ttagagacac cttttttgag ggcttggttg
     4441 atcctaatga aggagaagtt ggattggtta cgcctagtca gcagcatagt aatcattcag
     4501 aattggagca atcaccaata caactaaaag acttagaaag ccaaatacgg gatccaggtt
     4561 cctctgggtc ttccggttcc tcagggtcgt cggccaccat ggcttctaga atcctctggc
     4621 atgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg gaaaggaacg
     4681 tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg ggcccccaga
     4741 ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag gcccaagagt
     4801 ggtgcaggaa gtacatgaaa tcagggaatg tcaaggacct cacccaagcc tgggacctct
     4861 attatcatgt gttccgacga atctcaaaga ctagtggttc ctctgggtct tccggttcct
     4921 cagggtcgtc ggccaccatg gcttctagaa tcctctggca tgagatgtgg catgaaggcc
     4981 tggaagaggc atctcgtttg tactttgggg aaaggaacgt gaaaggcatg tttgaggtgc
     5041 tggagccctt gcatgctatg atggaacggg gcccccagac tctgaaggaa acatccttta
     5101 atcaggccta tggtcgagat ttaatggagg cccaagagtg gtgcaggaag tacatgaaat
     5161 cagggaatgt caaggacctc acccaagcct gggacctcta ttatcatgtg ttccgacgaa
     5221 tctcaaagac tagttatccg tacgacgtac cagactacgc ataaagcggc cgcgaggaga
     5281 tgaaacaaca tattcgaggc aattttggca atattatagt cattcaagag aaaacagtaa
     5341 atgtatgaaa aatggcaaga accttatctc tataggctta ataaatctgc ctttttggcc
     5401 aatttttcaa aacagatcct atttcttatt tatacacaat ctatatttaa cataattaag
     5461 tacgtgatga cattgaagat aataataagt aaagagttta cgtacttaaa ttttatatag
     5521 ttttagtata cttgcaatgt gtgaatggat ctagtaagct gaggtacttc tctagcgtga
     5581 atatgaaacg agtaacaggg tggtacataa tttacgaaca gaactcaagt tgataaatcc
     5641 tttgcgttct taaccatttg taaaagttcg tcaacaacat tgaaaatatt gcttcgagca
     5701 gatgttttat cgtttctgca aatttccagt cctgaacacc tctgttgtcg taaatgtaat
     5761 aatgccacac tggattcgat tctagaagct tggcgtaatc atggtcatag ctgtttcctg
     5821 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5881 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5941 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     6001 gaggcggttt gcgtattggg cgc
//
