LOCUS       sequence-473987-        6684 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DriD 3HB surface mutant used to interrogate the allosteric 
            mechanism.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6684)
  AUTHORS   Cannistraci E, Srinivasu BY, Chavez Orozco J Jr, Gozzi K, Wales TE, 
            Schumacher MA
  TITLE     Allosteric activation mechanism of DriD, a WYL-domain containing 
            transcription regulator.
  JOURNAL   Commun Biol. 2025 Apr 29;8(1):679. doi: 10.1038/s42003-025-08111-x.
  PUBMED    40301632
REFERENCE   2  (bases 1 to 6684)
  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: "Commun 
            Biol."; date: "2025-04-29"; volume: "8(1):679. doi"; pages: " 
            10.1038/s42003-025-08111-x"
COMMENT     Sequence Label: DriD-3HB-mutant
FEATURES             Location/Qualifiers
     source          1..6684
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(50..68)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      64..111
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(286..291,292..308,309..314))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(341..359)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        427..531
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(532..600,601..1392)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(750..769)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1563..2151
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2052..2071
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2305..2322
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2337..2479
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2405..2424)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2565..2587
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2581..2772)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4084..5166)
                     /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     5128..5147
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5167..5244)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5450..5469
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5553..5572
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5553..5571
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5572..5596
                     /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)."
     RBS             5611..5633
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5652..5669
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5679..5696
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
       61 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
      121 ggaactatat ccggatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc
      181 ctacagcatc cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatac
      241 acggtgcctg actgcgttag caatttaact gtgataaact accgcattaa agcttatcga
      301 tgataagctg tcaaacatga gaattcttga agacgaaagg gcctcgtgat acgcctattt
      361 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
      421 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
      481 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
      541 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
      601 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
      661 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
      721 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
      781 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
      841 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
      901 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
      961 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     1021 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     1081 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     1141 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     1201 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     1261 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     1321 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     1381 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     1441 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     1501 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     1561 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     1621 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     1681 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     1741 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     1801 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     1861 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     1921 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     1981 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     2041 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     2101 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     2161 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     2221 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     2281 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     2341 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatata tggtgcactc
     2401 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     2461 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     2521 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     2581 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     2641 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     2701 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     2761 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     2821 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     2881 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     2941 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     3001 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     3061 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     3121 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     3181 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     3241 ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat
     3301 ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc
     3361 tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag
     3421 gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg
     3481 cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg
     3541 atcagcggtc cagtgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt
     3601 ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg
     3661 ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc
     3721 agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg cttctcgccg
     3781 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     3841 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     3901 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     3961 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     4021 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     4081 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4141 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     4201 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     4261 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     4321 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     4381 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     4441 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     4501 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     4561 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     4621 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     4681 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     4741 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     4801 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     4861 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     4921 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     4981 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     5041 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     5101 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     5161 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     5221 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     5281 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     5341 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     5401 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     5461 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     5521 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     5581 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca
     5641 tgggcagcag ccatcatcat catcatcaca gcagcggcct ggtgccgcgc ggcagccata
     5701 tgcgtcacga gaaggcgacc cgtctgctgg atctggcgcg tatgctggcg ggtagcgcgg
     5761 aaggtctgac cctggatgaa atggcggcgg cgctgggtgt tggtcgtcgt accgcggagc
     5821 gtatgcgtga tgcggtgtgg gcggcgtttc cgcagatgga agcgatcgac gatccgccga
     5881 ccaagcgttt tcgtattccg agcggtctgg acggcgtttt ccaaaccccg accgcggagg
     5941 aactggcggc gctgcgtgtg gcggcggaca gcctgcgtgc gagcggtgcg gatgcgcgtg
     6001 cgagcagcct gtatgcgctg gaggcgaaac tgctgagcgc gctgcgtggc agcgcggcgg
     6061 aggaagtggc ggaggatgtt cgtgcgctgg tgcaggcgga aaccattgcg gttcatgcgg
     6121 gtccgcgtcc gtacgaagat caagcggtgc tgggtgcgat ccgtgcggcg attaaaggcc
     6181 tgcaagcgct gagctttcgt tatgagggtg gcagcacccc gggtcgtacc ccggaagtta
     6241 ccccgctggg tgtgctgttc ggccgtagca actacctggt tgcgctggaa ggcaagggtg
     6301 gcaaaccgcg tagctggcgt ctggaccgta tgagcgatct gaaagtgctg gacaaaccgg
     6361 cgccgccgcc gcaggatttc agcctgcaag cgtttgcgga cgagagcttc ggtatctatc
     6421 acgacgaaat tcaagatgtg gttctgcgta tccacaaaag ccgtgcggag gatgcgctgc
     6481 gttggcgttt ccacgcgacc cagcaagtta ccccggaagc ggacggtagc gtgctggtta
     6541 cctttcgtgc gggtggcatg cgtgagctga gctggcacct gttcacctgg ggcgatgcgg
     6601 tggaaattgt tgcgccgcag gttctgaaag acatgatggt gcaagaactg cgtgaagcgg
     6661 gtcgtgcgca tggtgcgtgg taag
//
