LOCUS       sequence-492057-        6276 bp    DNA     circular SYN 06-NOV-2025
DEFINITION  Insert an N-terminal FRB tag at VRG4 by popin-popout.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6276)
  TITLE     Glick lab plasmids 2025
REFERENCE   2  (bases 1 to 6276)
  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-FRB-Vrg4
FEATURES             Location/Qualifiers
     source          1..6276
                     /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"
     CDS             732..1010
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     primer_bind     complement(2774..2791)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2774..2790)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2783..2805)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2999..3018)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3118..3140
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3258..4061)
                     /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(4062..4277)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4284..4302)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4370..4474
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4475..4543,4544..5335)
                     /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(4693..4712)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5506..6094
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5995..6014
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6248..6265
                     /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 ccaagcttgt
      241 aatggaccat ggcaatgtgc gccatatata tatgcggctc ccttatgagt agaagaaaat
      301 atatgactgg tggtacaaag tggcatggtg gtatatagat gtaggaacac catttgagtt
      361 tgccataatg attaaatttc cctttttata aagaattagc cgcccataac acgaaaaata
      421 tcacagttga cgaaagaaga cacgtcgcct aaacgcgtaa tgaatcgcaa tatacgaaaa
      481 gctaaacggg tttttcagaa tcctcttacc cagccttgga taatagtttt ggccgaaaaa
      541 aaaaaaaaga aagaaaaaaa ttgaaatttc gcgatccgaa caaacaatga acagaaaaac
      601 agaatatata taacgctgcc aagcctattt cttgttgccg attaattgtt gacatcctct
      661 taattaccaa aagagcctaa gaaaacaaac acactaacca cacagtatct ttcgcccgaa
      721 tggcttctag aatcctctgg catgagatgt ggcatgaagg cctggaagag gcatctcgtt
      781 tgtactttgg ggaaaggaac gtgaaaggca tgtttgaggt gctggagccc ttgcatgcta
      841 tgatggaacg gggcccccag actctgaagg aaacatcctt taatcaggcc tatggtcgag
      901 atttaatgga ggcccaagag tggtgcagga agtacatgaa atcagggaat gtcaaggacc
      961 tcacccaagc ctgggacctc tattatcatg tgttccgacg aatctcaaag ggtggcagtg
     1021 gcggttcttc agaattgaaa acaggtcatg caggccataa cccttgggct tcagttgcca
     1081 attccggtcc gatctctatt ttatcctact gtggttcctc tattttaatg acggtgacta
     1141 acaagttcgt cgtcaatttg aaggatttca acatgaactt tgtcatgctt ttcgtgcaat
     1201 ctttggtttg tactataacc ttgattatcc tacgtatact gggctatgcg aagttccgtt
     1261 cattaaacaa aacagacgcc aagaactggt tccctatttc ctttttactg gtcttgatga
     1321 tctacacctc ttcgaaggct ttacaatact tggctgttcc aatttacacc attttcaaga
     1381 atttgactat tatcttgatt gcttatggtg aggttctctt ttttggtggc tctgtcacct
     1441 ccatggaatt gtcatcattt ttgttgatgg tcctttcttc tgtcgttgca acttggggtg
     1501 accagcaagc tgtggctgcc aaggctgctt cattggctga aggagcagcc ggtgctgttg
     1561 cctcctttaa cccaggttat ttctggatgt tcaccaactg tatcacttct gcattattcg
     1621 ttcttataat gagaaagaga attaagttaa ctaacttcaa ggatttcgac actatgtttt
     1681 acaacaatgt tttggctcta cctattctat tgctgttttc tttctgtgtg gaagattggt
     1741 cttcagttaa tttgaccaat aacttttcta acgattcgct aactgctatg atcatcagtg
     1801 gtgttgcatc cgtcggtatt tcttactgtt ccggttggtg tgttcgtgtt acttcgtcta
     1861 ctacatattc gatggtaggg gctttgaaca agctgccaat tgccttgtct ggtttgattt
     1921 tctttgatgc tccaagaaac ttcttatcta ttctctccat ttttattggt ttcctatcag
     1981 gtattattta tgctgttgcc aaacaaaaga agcaacaagc ccaaccttta cgtaaatgag
     2041 aacttacggg gggtgcaatt tatttttttt ttggtttatt ttattttata gaggcatcta
     2101 atgcaagtag atttatatac aattatactt aaaattgata taccttagaa caggtcggac
     2161 acaacgtgtg gtaactccaa tatatcacta atgacaatag cgccttctgg tgtttgcccc
     2221 aaaatttcat ttacttcatt ctcaactaaa tgaatacatg ttttcatgcc caatttgata
     2281 cctgtctcga tgtttttccc actgtcatct ataaaataag cattctcgta tcttgccaac
     2341 ccgctttcct tcatggcttt ttcaaatgct ttaacatgtg gtttgcagac cagcgtatct
     2401 gtcctagagt agtcacaata tgtcaatcca tcaaataaat ccgctatacc caacaatctc
     2461 aggcaacgta ttgcgtggtt tttataagcg ttggtgaaaa gccacaattt atcaatcttt
     2521 cccgattgtc tcaatctcaa cagcatattc cttaatggta tatcaggttt caagatatct
     2581 tgtagtggta aggaatcatc cacaagccgg ttatattcca gtgcgttcac tttgtggaac
     2641 attacaagtc cccttatggc aagtccatac tctttgtagt aagagttatt caaaacgtga
     2701 gcatcttcag gtgatagttt caagtgtgtt tgaaagaacc tcagtataga ttgttgcata
     2761 agatcgtgaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     2821 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     2881 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc
     2941 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta
     3001 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     3061 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3121 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcg
     3181 ggtaataact gatataatta aattgaagct ctaatttgtg agtttagtat acatgcattt
     3241 acttataata cagtttttta gttttgctgg ccgcatcttc tcaaatatgc ttcccagcct
     3301 gcttttctgt aacgttcacc ctctacctta gcatcccttc cctttgcaaa tagtcctctt
     3361 ccaacaataa taatgtcaga tcctgtagag accacatcat ccacggttct atactgttga
     3421 cccaatgcgt ctcccttgtc atctaaaccc acaccgggtg tcataatcaa ccaatcgtaa
     3481 ccttcatctc ttccacccat gtctctttga gcaataaagc cgataacaaa atctttgtcg
     3541 ctcttcgcaa tgtcaacagt acccttagta tattctccag tagataggga gcccttgcat
     3601 gacaattctg ctaacatcaa aaggcctcta ggttcctttg ttacttcttc tgccgcctgc
     3661 ttcaaaccgc taacaatacc tgggcccacc acaccgtgtg cattcgtaat gtctgcccat
     3721 tctgctattc tgtatacacc cgcagagtac tgcaatttga ctgtattacc aatgtcagca
     3781 aattttctgt cttcgaagag taaaaaattg tacttggcgg ataatgcctt tagcggctta
     3841 actgtgccct ccatggaaaa atcagtcaag atatccacat gtgtttttag taaacaaatt
     3901 ttgggaccta atgcttcaac taactccagt aattccttgg tggtacgaac atccaatgaa
     3961 gcacacaagt ttgtttgctt ttcgtgcatg atattaaata gcttggcagc aacaggacta
     4021 ggatgagtag cagcacgttc cttatatgta gctttcgaca tgatttatct tcgtttcctg
     4081 catgtttttg ttctgtgcag ttgggttaag aatactgggc aatttcatgt ttcttcaaca
     4141 ctacatatgc gtatatatac caatctaagt ctgtgctcct tccttcgttc ttccttctgt
     4201 tcggagatta ccgaatcaaa aaaatttcaa ggaaaccgaa atcaaaaaaa agaataaaaa
     4261 aaaaatgatg aattgaaaag ctggcctcgt gatacgccta tttttatagg ttaatgtcat
     4321 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4381 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4441 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     4501 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     4561 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     4621 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     4681 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     4741 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     4801 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     4861 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     4921 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     4981 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     5041 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     5101 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     5161 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     5221 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     5281 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     5341 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5401 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     5461 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5521 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5581 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5641 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5701 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     5761 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     5821 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     5881 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     5941 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6001 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6061 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6121 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     6181 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     6241 cgagcgcagc gagtcagtga gcgaggaagc ggaaga
//
