LOCUS       sequence-226757-        4446 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTet-W108 scRNA.b2, pTet-RR2-sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4446)
  AUTHORS   Dong C, Fontana J, Patel A, Carothers JM, Zalatan JG
  TITLE     Synthetic CRISPR-Cas gene activators for transcriptional 
            reprogramming in bacteria.
  JOURNAL   Nat Commun. 2018 Jun 27;9(1):2489. doi: 10.1038/s41467-018-04901-6.
  PUBMED    29950558
REFERENCE   2  (bases 1 to 4446)
  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: "2018-06-27"; volume: "9(1):2489. doi"; pages: " 
            10.1038/s41467-018-04901-6"
COMMENT     Sequence Label: pJF131B
FEATURES             Location/Qualifiers
     source          1..4446
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     488..507
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(726..830)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     898..916
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(920..963)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             complement(981..1607)
                     /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"
     promoter        1623..1678
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    1659..1677
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        1770..1788
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     CDS             1811..1840
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1856..1873
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2099..2145
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2180..2806)
                     /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"
     promoter        2822..2877
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    2858..2876
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        2907..2982
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             3003..3032
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3048..3065
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3291..3337
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3381..3398)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3552..4140)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3632..3651)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4311..4446,1..656,657..725))
                     /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"
ORIGIN
        1 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
       61 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
      121 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
      181 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
      241 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
      301 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
      361 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
      421 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
      481 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
      541 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
      601 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
      661 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
      721 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
      781 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
      841 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
      901 aggcgtatca cgaggcagaa tttcagataa aaaaaatcct tagctttcgc taaggatgat
      961 ttctggaatt ctaaagatct ttaagaccca ctttcacatt taagttgttt ttctaatccg
     1021 catatgatca attcaaggcc gaataagaag gctggctctg caccttggtg atcaaataat
     1081 tcgatagctt gtcgtaataa tggcggcata ctatcagtag taggtgtttc cctttcttct
     1141 ttagcgactt gatgctcttg atcttccaat acgcaaccta aagtaaaatg ccccacagcg
     1201 ctgagtgcat ataatgcatt ctctagtgaa aaaccttgtt ggcataaaaa ggctaattga
     1261 ttttcgagag tttcatactg tttttctgta ggccgtgtac ctaaatgtac ttttgctcca
     1321 tcgcgatgac ttagtaaagc acatctaaaa cttttagcgt tattacgtaa aaaatcttgc
     1381 cagctttccc cttctaaagg gcaaaagtga gtatggtgcc tatctaacat ctcaatggct
     1441 aaggcgtcga gcaaagcccg cttatttttt acatgccaat acaatgtagg ctgctctaca
     1501 cctagcttct gggcgagttt acgggttgtt aaaccttcga ttccgacctc attaagcagc
     1561 tctaatgcgc tgttaatcac tttactttta tctaatctag acatcattaa ttcctaattt
     1621 ttgttgacac tctatcgttg atagagttat tttaccactc cctatcagtg atagagaaaa
     1681 actagtgaag atccggcctg cagccagttt tagagctaga aatagcaagt taaaataagg
     1741 ctagtccgtt atcaacttga aaaagtggca catgaggatc acccatgtgc tttttttgaa
     1801 gcttgggccc gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca
     1861 tcatcatcat cattgagttt aaacggtctc cagcttggct gttttggcgg atgagagaag
     1921 attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg
     1981 cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc
     2041 cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca
     2101 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     2161 gaactggatc cttactcgat taagacccac tttcacattt aagttgtttt tctaatccgc
     2221 atatgatcaa ttcaaggccg aataagaagg ctggctctgc accttggtga tcaaataatt
     2281 cgatagcttg tcgtaataat ggcggcatac tatcagtagt aggtgtttcc ctttcttctt
     2341 tagcgacttg atgctcttga tcttccaata cgcaacctaa agtaaaatgc cccacagcgc
     2401 tgagtgcata taatgcattc tctagtgaaa aaccttgttg gcataaaaag gctaattgat
     2461 tttcgagagt ttcatactgt ttttctgtag gccgtgtacc taaatgtact tttgctccat
     2521 cgcgatgact tagtaaagca catctaaaac ttttagcgtt attacgtaaa aaatcttgcc
     2581 agctttcccc ttctaaaggg caaaagtgag tatggtgcct atctaacatc tcaatggcta
     2641 aggcgtcgag caaagcccgc ttatttttta catgccaata caatgtaggc tgctctacac
     2701 ctagcttctg ggcgagttta cgggttgtta aaccttcgat tccgacctca ttaagcagct
     2761 ctaatgcgct gttaatcact ttacttttat ctaatctaga catcattaat tcctaatttt
     2821 tgttgacact ctatcgttga tagagttatt ttaccactcc ctatcagtga tagagaaaag
     2881 actagttgga accgtactgg aactgcgttt tagagctaga aatagcaagt taaaataagg
     2941 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg aagcttgggc
     3001 ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat catcatcatc
     3061 atcattgagt ttaaacggtc tccagcttgg ctgttttggc ggatgagaga agattttcag
     3121 cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg
     3181 cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc
     3241 cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat caaataaaac
     3301 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaactctc
     3361 gagtctagac tgcaggcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3421 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3481 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3541 ttgctggcgt ttttccacag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3601 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3661 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3721 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3781 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3841 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3901 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3961 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     4021 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4081 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4141 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4201 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4261 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4321 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4381 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4441 atgata
//
