LOCUS       sequence-327739-        4314 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LacI protein regulates expression of yellow fluorescent protein. For
            measuring dose-response curves..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4314)
  AUTHORS   Tack DS, Tonner PD, Pressman A, Olson ND, Levy SF, Romantseva EF, 
            Alperovich N, Vasilyeva O, Ross D
  TITLE     The genotype-phenotype landscape of an allosteric protein.
  JOURNAL   Mol Syst Biol. 2021 Mar;17(3):e10179. doi: 10.15252/msb.202010179.
  PUBMED    33784029
REFERENCE   2  (bases 1 to 4314)
  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"; journalName: "Mol Syst 
            Biol."; date: "2021-03"; volume: "17(3):e10179. doi"; pages: " 
            10.15252/msb.202010179"
COMMENT     Sequence Label: pVER-LacI
FEATURES             Location/Qualifiers
     source          1..4314
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(84..103)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             95..910
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             95..910
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(168..187)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      999..1544
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     1445..1464
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1909..1928
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(2129..3211)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3173..3192
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             complement(join(3359..4072,4073..4075,4076..4078))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3404..3425)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3753..3772
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4012..4033
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_RNA        complement(4128..4177)
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     protein_bind    4182..4198
                     /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(4206..4212,4213..4228,4229..4234))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
ORIGIN
        1 ctcaaaatct ctgatgtaca ttgcacaaga taaaaatata tcatcatgaa caataaaact
       61 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
      121 ttgctccagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
      181 tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc
      241 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
      301 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
      361 tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt
      421 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
      481 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
      541 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
      601 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataagcttt tgccattctc
      661 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
      721 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
      781 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
      841 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
      901 gtttttctaa ggtaactgtc agaccaagtt tactcatata tactttagat tgatttttcg
      961 ttccactgag cgtcagaccc cttaataaga tgatcttctt gagatcgttt tggtctgcgc
     1021 gtaatctctt gctctgaaaa cgaaaaaacc gccttgcagg gcggtttttc gaaggttctc
     1081 tgagctacca actctttgaa ccgaggtaac tggcttggag gagcgcagtc accaaaactt
     1141 gtcctttcag tttagcctta accggcgcat gacttcaaga ctaactcctc taaatcaatt
     1201 accagtggct gctgccagtg gtgcttttgc atgtctttcc gggttggact caagacgata
     1261 gttaccggat aaggcgcagc ggtcggactg aacggggggt tcgtgcatac agtccagctt
     1321 ggagcgaact gcctacccgg aactgagtgt caggcgtgga atgagacaaa cgcggccata
     1381 acagcggaat gacaccggta aaccgaaagg caggaacagg agagcgcacg agggagccgc
     1441 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccaccac tgatttgagc
     1501 gtcagatttc gtgatgcttg tcaggggggc ggagcctatg gaaaaacggc tttgccgcgg
     1561 ccctctcact tccctgttaa gtatcttcct ggcatcttcc aggaaatctc cgccccgttc
     1621 gtaagccatt tccgctcgcc gcagtcgaac gaccgagcgt agcgagtcag tgagcgagga
     1681 agcggaatat atcctgtatc acatattctg ctgacgcacc ggtgcagcct tttttctcct
     1741 gccacatgaa gcacttcact gacaccctca tcagtgccaa catagtaagc cagtatacac
     1801 tccgctagcg ctgaggtctg cctcgtgcag cgagtcagtg agcgaggaag cacctcagat
     1861 aaaatatttg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     1921 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     1981 gtagaggatc tgctcatgtt tgacagctta tcatcgatgc ataatgtgcc tgtcaaatgg
     2041 acgaagcagg gattctgcaa accctatgct actccctcga gccgtcaatt gtctgattcg
     2101 ttaccaatta ttttttcctc ctggattatc actgcccgct ttccagtcgg gaaacctgtc
     2161 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     2221 ccagggtggt ttttcttttc accagtgaga ctggcaacag ctgattgccc ttcaccgcct
     2281 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     2341 gtttgatggt ggttaacggc gggatataac atgagctatc ttcggtatcg tcgtatccca
     2401 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     2461 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     2521 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     2581 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     2641 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     2701 cgcccagtcg cgtaccgtcc tcatgggaga aaataatact gttgatgggt gtctggtcag
     2761 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     2821 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     2881 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     2941 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     3001 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     3061 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     3121 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     3181 ctgcgacatc gtataacgtt actggtttca tattcaccac cctgaattga ctctcttccg
     3241 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggcgcgc cgccattgga
     3301 ccaaaacgaa aaaaggcccc cctttcggga ggcctctttt ctggaatttg gtaccgagtt
     3361 acttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga
     3421 ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactggtag ctaaggtagt
     3481 ggttgtcggg cagcagcacg gggccgtcgc cgattggggt gttctgctgg tagtggtcgg
     3541 cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt
     3601 tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt
     3661 gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca
     3721 gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga
     3781 agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgcagct
     3841 tcatgtggtc ggggtagcgg gcgaagcatt gcaggccgta gccgaaggtg gtcacgaggg
     3901 tgggccaggg cacgggcagc ttgcctgtgg tgcagatgaa cttcagggtc agcttgccgt
     3961 aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt
     4021 ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccatct
     4081 agtatttccc ctctttctct agtattaaac aaaattattt gtagaggctg tttcgtcctc
     4141 acggactcat cagaccggaa agcacatccg gtgacagcta attgttatcc gctcacaatt
     4201 ccacacatta tacgagccga tgattaattg tcaacacagc caacgatcgt taagcgggag
     4261 accagaaaca aaaaaaggcc ccccgttagg gaggccttca ataattggtt gtgt
//
