LOCUS       sequence-233520-        3205 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  aTc inducible gRNA expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3205)
  AUTHORS   Zhang J, Poh CL
  TITLE     Regulating exopolysaccharide gene wcaF allows control of Escherichia
            coli biofilm formation.
  JOURNAL   Sci Rep. 2018 Sep 3;8(1):13127. doi: 10.1038/s41598-018-31161-7.
  PUBMED    30177768
REFERENCE   2  (bases 1 to 3205)
  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: "Sci Rep."; 
            date: "2018-09-3"; volume: "8(1):13127. doi"; pages: " 
            10.1038/s41598-018-31161-7"
COMMENT     Sequence Label: pBbE2k-pTet-gRNAwcaF159
FEATURES             Location/Qualifiers
     source          1..3205
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     108..127
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(324..950)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     855..874
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        966..1021
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    1002..1020
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        1059..1134
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1155..1184
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1200..1217
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1273..1290)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1443..1489
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1534..1605
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1621..1648
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(1844..2432)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1924..1943)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2467..2561)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(2592..3205,1..181))
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2703..2722)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg
       61 ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc cagtcatagc
      121 cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct tgttcaatca
      181 tgcgaaacga tcctcatcct gtctcttgat cagatcatga tcccctgcgc catcagatcc
      241 ttggcggcaa gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg
      301 ccccagctgg caattccgac gtcttaagac ccactttcac atttaagttg tttttctaat
      361 ccgcatatga tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat
      421 aattcgatag cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct
      481 tctttagcga cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca
      541 gcgctgagtg catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat
      601 tgattttcga gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct
      661 ccatcgcgat gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct
      721 tgccagcttt ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg
      781 gctaaggcgt cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct
      841 acacctagct tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc
      901 agctctaatg cgctgttaat cactttactt ttatctaatc tagacatcat taattcctaa
      961 tttttgttga cactctatcg ttgatagagt tattttacca ctccctatca gtgatagaga
     1021 aaagaattca aaagatctcg tttattcgga gcaaaaatgt tttagagcta gaaatagcaa
     1081 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     1141 tgaagcttgg gcccgaacaa aaactcatct cagaagagga tctgaatagc gccgtcgacc
     1201 atcatcatca tcatcattga gtttaaacgg tctccagctt ggctgttttg gcggatgaga
     1261 gaagattttc agcctgatac agattaaatc agaacgcaga agcggtctga taaaacagaa
     1321 tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa
     1381 acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc
     1441 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     1501 cggatccaaa ctcgagtaag gatctccagg catcaaataa aacgaaaggc tcagtcgaaa
     1561 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta gagtcacact
     1621 ggctcacctt cgggtgggcc tttctgcgtt tatacctagg gcgttcggct gcggcgagcg
     1681 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     1741 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaagtga gcaaaaggcc
     1801 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     1861 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     1921 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     1981 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2041 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2101 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2161 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     2221 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     2281 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     2341 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     2401 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     2461 gcttggattc tcaccaataa aaaacgcccg gcggcaaccg agcgttctga acaaatccag
     2521 atggagttct gaggtcatta ctggatctat caacaggagt ccaagcgagc tctcgaaccc
     2581 cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg
     2641 ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca
     2701 gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca
     2761 cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg
     2821 ccatgggtca cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt
     2881 tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct
     2941 tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta
     3001 gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca
     3061 ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc
     3121 cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc
     3181 cacgatagcc gcgctgcctc gtcct
//
