LOCUS       sequence-201393-        4250 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pUC-19 derivative reporter plasmid for the screening of 
            Transcription Factors (TFs) carrying the DNA-binding domain of LacI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4250)
  AUTHORS   Juarez JF, Lecube-Azpeitia B, Brown SL, Johnston CD, Church GM
  TITLE     Biosensor libraries harness large classes of binding domains for 
            construction of allosteric transcriptional regulators.
  JOURNAL   Nat Commun. 2018 Aug 6;9(1):3101. doi: 10.1038/s41467-018-05525-6.
  PUBMED    30082754
REFERENCE   2  (bases 1 to 4250)
  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: "Nat 
            Commun."; date: "2018-08-6"; volume: "9(1):3101. doi"; pages: " 
            10.1038/s41467-018-05525-6"
COMMENT     Sequence Label: pHC_DYOLacI-R
FEATURES             Location/Qualifiers
     source          1..4250
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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     42..64
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     RBS             75..83
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             89..802
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGFVTILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     900..922
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(960..978)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1046..1150
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1151..1219,1220..2011)
                     /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(1369..1388)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2182..2770
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2671..2690
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2924..2941
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3058..3079
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        complement(3796..3873)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     3985..4004
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacat gacctaagga ggtaaataat gcgtaaaggc gaagagctgt tcactggttt
      121 cgtcactatt ctggtggaac tggatggtga tgtcaacggt cataagtttt ccgtgcgtgg
      181 cgagggtgaa ggtgacgcaa ctaatggtaa actgacgctg aagttcatct gtactactgg
      241 taaactgccg gtaccttggc cgactctggt aacgacgctg acttatggtg ttcagtgctt
      301 tgctcgttat ccggaccaca tgaagcagca tgacttcttc aagtccgcca tgccggaagg
      361 ctatgtgcag gaacgcacga tttcctttaa ggatgacggc acgtacaaaa cgcgtgcgga
      421 agtgaaattt gaaggcgata ccctggtaaa ccgcattgag ctgaaaggca ttgactttaa
      481 agaagacggc aatatcctgg gccataagct ggaatacaat tttaacagcc acaatgttta
      541 catcaccgcc gataaacaaa aaaatggcat taaagcgaat tttaaaattc gccacaacgt
      601 ggaggatggc agcgtgcagc tggctgatca ctaccagcaa aacactccaa tcggtgatgg
      661 tcctgttctg ctgccagaca atcactatct gagcacgcaa agcgttctgt ctaaagatcc
      721 gaacgagaaa cgcgatcaca tggttctgct ggagttcgta accgcagcgg gcatcacgca
      781 tggtatggat gaactgtaca aataattaag ccagccccga cacccgccaa cacccgctga
      841 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
      901 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
      961 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     1021 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1081 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     1141 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     1201 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     1261 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     1321 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     1381 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     1441 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     1501 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     1561 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     1621 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     1681 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     1741 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     1801 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     1861 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     1921 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     1981 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     2041 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     2101 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2161 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2221 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2281 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     2341 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2401 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2461 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     2521 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2581 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     2641 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     2701 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     2761 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     2821 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     2881 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     2941 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     3001 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     3061 tgtgagttag ctcactcatt aggcttattt gtacagctca tccatgccac cggtagaatg
     3121 acgaccctcc gcgcgctcat attgctctac gatcgtataa tcttcattat gagaggtgat
     3181 gtccagttta atattcacat tgtacgcgcc aggcagctgc acaggtttct tggctttgta
     3241 cgtggttttc acttcagcgt cataatggcc gccatctttc agtttcaggc gctgtttaat
     3301 ttcgcctttc agcgcaccat cttccggata catgcgttcg ctagacgcct cccaacccat
     3361 cgtctttttc tgcatcaccg ggccatcaga tggaaaatta gtaccacgca gtttaacttt
     3421 atagatgaac tcgccatcct gcagggagga gtcctgagtg acggtcacga caccaccatc
     3481 ttcaaaattc attacgcgtt cccacttgaa accttccgga aaagacagct tcagatagtc
     3541 cgggatatcc gctgggtgtt taacatacgc tttagaaccg tacataaatt gcgggctcag
     3601 gatgtcccac gcaaaaggca gcgggccgcc tttagtcact ttcagtttgg cggtctgggt
     3661 gccttcatac ggacggccct cgccttcgcc ttcgatctcg aactcgtgac cgttaacaga
     3721 accctccatg tgaactttga agcgcatgaa ctctttaatg atagccatgt tatcctcctc
     3781 gcccttggaa accatattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     3841 accgcgaaag gttttgcacc attcgatggt gtcgggtacc aaggagatgg cgcccaacag
     3901 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     3961 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     4021 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatccggg cttatcgact
     4081 gcacggtgca ccaatgcttc tggcgtcagg cagccatcgg aagctgtggt atggctgtgc
     4141 aggtcgtaaa tcactgcata attcgtgtcg ctcaaggcgc actcccgttc tggataatgt
     4201 tttttgcgcc gacatcataa cggttctggc aaatattctg aaatgagctg
//
