LOCUS       pKDsgRNA-FRT                 6928 bp    DNA     circular     30-SEP-2021
DEFINITION  sgRNA-FRT top strand in pKDsgRNA-NT SpecR 28C.
SOURCE      
  ORGANISM  
COMMENT     AUTHORNAME|Schnetz|
COMMENT     AUTHOREML|schnetz@uni-koeln.de|
FEATURES             Location/Qualifiers
     promoter        5301..5329
                     /vntifkey="30"
                     /label=P-tet
                     /note="tet promoter (-35 and -10) "
     protein_bind    5307..5325
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /vntifkey="31"
                     /label=tet\operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5282..5300
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /vntifkey="31"
                     /label=tet\operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     rep_origin      4494..4716
                     /vntifkey="33"
                     /label=pSC101\ori
                     /note="low-copy replication origin that requires the Rep101 protein"
     CDS             complement(3496..4446)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /vntifkey="4"
                     /label=Rep101(Ts)
     terminator      3129..3373
                     /vntifkey="43"
                     /label=-lambda\tL3\terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             2448..3128
                     /codon_start=1
                     /gene="exo"
                     /product="5' to 3' double-stranded DNA exonuclease in the
                     lambda Red system"
                     /vntifkey="4"
                     /label=exo
                     /note="creates 3' single-stranded DNA tails"
     CDS             1666..2451
                     /codon_start=1
                     /gene="bet"
                     /product="single-stranded DNA binding recombinase in the
                     lambda Red system"
                     /vntifkey="4"
                     /label=Beta
                     /note="mediates pairing of a single-stranded DNA with a complementary target"
     CDS             1244..1660
                     /codon_start=1
                     /gene="gam"
                     /product="inhibitor of the host RecBCD nuclease in the
                     lambda Red system"
                     /vntifkey="4"
                     /label=gam
                     /note="protects the double-stranded DNA substrate for recombination"
     promoter        916..1200
                     /gene="araBAD"
                     /vntifkey="30"
                     /label=araBAD\promoter
                     /note="promoter of the L-arabinose operon of E. coli; the araC regulatory gene is transcribed in the opposite direction (Guzman et al., 1995)"
     primer_bind     555..564
                     /vntifkey="28"
                     /label=M13\Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward"
     CDS             complement(11..889)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /vntifkey="4"
                     /label=araC
     promoter        complement(6717..6808)
                     /gene="bla"
                     /vntifkey="30"
                     /label=AmpR\promoter
     CDS             complement(5925..6716)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy,
                     1985)"
                     /vntifkey="4"
                     /label=SmR
                     /note="confers resistance to spectinomycin and streptomycin"
     terminator      5740..5786
                     /vntifkey="43"
                     /label=rrnB\T1\terminator
                     /note="transcription terminator T1 from the E. coli rrnB gene"
     CDS             5497..5514
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /vntifkey="4"
                     /label=6xHis
     CDS             5452..5481
                     /codon_start=1
                     /product="Myc (human c-Myc oncogene) epitope tag"
                     /vntifkey="4"
                     /label=Myc
     misc_RNA        5356..5431
                     /vntifkey="53"
                     /label=g\RNA\scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system"
     misc_RNA        5336..5431
                     /vntifkey="53"
                     /label=sgRNA-FRT
BASE COUNT     1828 a      1640 c      1597 g      1863 t 
ORIGIN
        1 catcgattta ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac 
       61 ggaactcgct cgggctggcc ccggtgcatt ttttaaatac ccgcgagaag tagagttgat 
      121 cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca 
      181 gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct 
      241 ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga 
      301 tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat 
      361 tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct 
      421 caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga 
      481 tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg 
      541 tattggcaaa tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt 
      601 aaacccactg gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc 
      661 ctggcgggaa cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca 
      721 ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt 
      781 cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg 
      841 cattaaacga gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac 
      901 tcccgccatt cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg 
      961 tcttttactg gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt 
     1021 aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca 
     1081 gaaaagtcca cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat 
     1141 ccataagatt agcggatcct acctgacgct ttttatcgca actctctact gtttctccat 
     1201 acccgttttt ttgggaattc gagctctaag gaggttataa aaaatggata ttaatactga 
     1261 aactgagatc aagcaaaagc attcactaac cccctttcct gttttcctaa tcagcccggc 
     1321 atttcgcggg cgatattttc acagctattt caggagttca gccatgaacg cttattacat 
     1381 tcaggatcgt cttgaggctc agagctgggc gcgtcactac cagcagctcg cccgtgaaga 
     1441 gaaagaggca gaactggcag acgacatgga aaaaggcctg ccccagcacc tgtttgaatc 
     1501 gctatgcatc gatcatttgc aacgccacgg ggccagcaaa aaatccatta cccgtgcgtt 
     1561 tgatgacgat gttgagtttc aggagcgcat ggcagaacac atccggtaca tggttgaaac 
     1621 cattgctcac caccaggttg atattgattc agaggtataa aacgaatgag tactgcactc 
     1681 gcaacgctgg ctgggaagct ggctgaacgt gtcggcatgg attctgtcga cccacaggaa 
     1741 ctgatcacca ctcttcgcca gacggcattt aaaggtgatg ccagcgatgc gcagttcatc 
     1801 gcattactga tcgttgccaa ccagtacggc cttaatccgt ggacgaaaga aatttacgcc 
     1861 tttcctgata agcagaatgg catcgttccg gtggtgggcg ttgatggctg gtcccgcatc 
     1921 atcaatgaaa accagcagtt tgatggcatg gactttgagc aggacaatga atcctgtaca 
     1981 tgccggattt accgcaagga ccgtaatcat ccgatctgcg ttaccgaatg gatggatgaa 
     2041 tgccgccgcg aaccattcaa aactcgcgaa ggcagagaaa tcacggggcc gtggcagtcg 
     2101 catcccaaac ggatgttacg tcataaagcc atgattcagt gtgcccgtct ggccttcgga 
     2161 tttgctggta tctatgacaa ggatgaagcc gagcgcattg tcgaaaatac tgcatacact 
     2221 gcagaacgtc agccggaacg cgacatcact ccggttaacg atgaaaccat gcaggagatt 
     2281 aacactctgc tgatcgccct ggataaaaca tgggatgacg acttattgcc gctctgttcc 
     2341 cagatatttc gccgcgacat tcgtgcatcg tcagaactga cacaggccga agcagtaaaa 
     2401 gctcttggat tcctgaaaca gaaagccgca gagcagaagg tggcagcatg acaccggaca 
     2461 ttatcctgca gcgtaccggg atcgatgtga gagctgtcga acagggggat gatgcgtggc 
     2521 acaaattacg gctcggcgtc atcaccgctt cagaagttca caacgtgata gcaaaacccc 
     2581 gctccggaaa gaagtggcct gacatgaaaa tgtcctactt ccacaccctg cttgctgagg 
     2641 tttgcaccgg tgtggctccg gaagttaacg ctaaagcact ggcctgggga aaacagtacg 
     2701 agaacgacgc cagaaccctg tttgaattca cttccggcgt gaatgttact gaatccccga 
     2761 tcatctatcg cgacgaaagt atgcgtaccg cctgctctcc cgatggttta tgcagtgacg 
     2821 gcaacggcct tgaactgaaa tgcccgttta cctcccggga tttcatgaag ttccggctcg 
     2881 gtggtttcga ggccataaag tcagcttaca tggcccaggt gcagtacagc atgtgggtga 
     2941 cgcgaaaaaa tgcctggtac tttgccaact atgacccgcg tatgaagcgt gaaggcctgc 
     3001 attatgtcgt gattgagcgg gatgaaaagt acatggcgag ttttgacgag atcgtgccgg 
     3061 agttcatcga aaaaatggac gaggcactgg ctgaaattgg ttttgtattt ggggagcaat 
     3121 ggcgatgacg catcctcacg ataatatccg ggtaggcgca atcactttcg tctactccgt 
     3181 tacaaagcga ggctgggtat ttcccggcct ttctgttatc cgaaatccac tgaaagcaca 
     3241 gcggctggct gaggagataa ataataaacg aggggctgta tgcacaaagc atcttctgtt 
     3301 gagttaagaa cgagtatcga gatggcacat agccttgctc aaattggaat caggtttgtg 
     3361 ccaataccag tagaaacaga cgaagaatcc atgggtatgg acagttttcc ctttgatatg 
     3421 taacggtgaa cagttgttct acttttgttt gttagtcttg atgcttcact gatagataca 
     3481 agagccataa gaacctcaga tccttccgta tttagccagt atgttctcta gtgtggttcg 
     3541 ttgtttttgc gtgagccatg agaacgaacc attgagatca tacttacttt gcatgtcact 
     3601 caaaaatttt gcctcaaaac tggtgagctg aatttttgca gttaaagcat cgtgtagtgt 
     3661 ttttcttagt ccgttatgta ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc 
     3721 attcattttt atctggttgt tctcaagttc ggttacgaga tccatttgtc tatctagttc 
     3781 aacttggaaa atcaacgtat cagtcgggcg gcctcgctta tcaaccacca atttcatatt 
     3841 gctgtaagtg tttaaatctt tacttattgg tttcaaaacc cattggttaa gccttttaaa 
     3901 ctcatggtag ttattttcaa gcattaacat gaacttaaat tcatcaaggc taatctctat 
     3961 atttgccttg tgagttttct tttgtgttag ttcttttaat aaccactcat aaatcctcat 
     4021 agagtatttg ttttcaaaag acttaacatg ttccagatta tattttatga atttttttaa 
     4081 ctggaaaaga taaggcaata tctcttcact aaaaactaat tctaattttt cgcttgagaa 
     4141 cttggcatag tttgtccact ggaaaatctc aaagccttta accaaaggat tcctgatttc 
     4201 cacagttctc gtcatcagct ctctggttgc tttagctaat acaccataag cattttccct 
     4261 actgatgttc atcatctgaa cgtattggtt ataagtgaac gataccgtcc gttctttcct 
     4321 tgtagggttt tcaatcgtgg ggttgagtag tgccacacag cataaaatta gcttggtttc 
     4381 atgctccgtt aagtcatagc gactaatcgc tagttcattt gctttgaaaa caactaattc 
     4441 agacatacat ctcaattggt ctaggtgatt ttaatcacta taccaattga gatgggctag 
     4501 tcaatgataa ttactagtcc ttttcctttg agttgtgggt atctgtaaat tctgctagac 
     4561 ctttgctgga aaacttgtaa attctgctag accctctgta aattccgcta gacctttgtg 
     4621 tgtttttttt gtttatattc aagtggttat aatttataga ataaagaaag aataaaaaaa 
     4681 gataaaaaga atagatccca gccctgtgta taactcacta ctttagtcag ttccgcagta 
     4741 ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt aaaaccctaa 
     4801 aggcttaagt agcaccctcg caagctcggt tgcggccgca atcgggcaaa tcgctgaata 
     4861 ttccttttgt ctccgaccat caggcacctg agtcgctgtc tttttcgtga cattcagttc 
     4921 gctgcgctca cggctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg 
     4981 attcatgcaa ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt 
     5041 tttatggcgg gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc 
     5101 tctgattttc cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa 
     5161 tgcacccagt aaggcagcgg tatcatcaac ggggtctgac gctcagtgga acgaaaactc 
     5221 acgttaaggg attttggtca tgagattatc agctttcgct aaggatgatt tctggaattc 
     5281 ttccctatca gtgatagaga ttgacatccc tatcagtgat agagatactg agcactccta 
     5341 tactttctag agaatgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta 
     5401 tcaacttgaa aaagtggcac cgagtcggtg ctttttttga agcttgggcc cgaacaaaaa 
     5461 ctcatctcag aagaggatct gaatagcgcc gtcgaccatc atcatcatca tcattgagtt 
     5521 taaacggtct ccagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga 
     5581 ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg 
     5641 tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg 
     5701 tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag 
     5761 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaactggat ccttactcga 
     5821 gtctagactg caggcggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 
     5881 caatctaaag tatatatgag taaacttggt ctgctgacag gacattattt gccgactacc 
     5941 ttggtgatct cgcctttcac gtagtggaca aattcttcca actgatctgc gcgcgaggcc 
     6001 aagcgatctt cttcttgtcc aagataagcc tgtctagctt caagtatgac gggctgatac 
     6061 tgggccggca ggcgctccat tgcccagtcg gcagcgacat ccttcggcgc gattttgccg 
     6121 gttactgcgc tgtaccaaat gcgggacaac gtaagcacta catttcgctc atcgccagcc 
     6181 cagtcgggcg gcgagttcca tagcgttaag gtttcattta gcgcctcaaa tagatcctgt 
     6241 tcaggaaccg gatcaaagag ttcctccgcc gctggaccta ccaaggcaac gctatgttct 
     6301 cttgcttttg tcagcaagat agccagatca atgtcgatcg tggctggctc gaagatacct 
     6361 gcaagaatgt cattgcgctg ccattctcca aattgcagtt cgcgcttagc tggataacgc 
     6421 cacggaatga tgtcgtcgtg cacaacaatg gtgacttcta cagcgcggag aatctcgctc 
     6481 tctccagggg aagccgaagt ttccaaaagg tcgttgatca aagctcgccg cgttgtttca 
     6541 tcaagcctta cggtcaccgt aaccagcaaa tcaatatcac tgtgtggctt caggccgcca 
     6601 tccactgcgg agccgtacaa atgtacggcc agcaacgtcg gttcgagatg gcgctcgatg 
     6661 acgccaacta cctctgatag ttgagtcgat acttcggcga tcaccgcttc cctcatactc 
     6721 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 
     6781 tttgaatgta tttagaaaaa taaacaaata gctagctcac tcggtcgcta ccagggttat 
     6841 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 
     6901 cgcacatttc cccgaaaagt gccacctg 
//
