LOCUS       sequence-202037-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains arabinose-induced lambda Red and a tet-inducible gRNA 
            targeting pfkB..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6928)
  AUTHORS   Moreb EA, Hoover B, Yaseen A, Valyasevi N, Roecker Z, Menacho-Melgar
            R, Lynch MD
  TITLE     Managing the SOS Response for Enhanced CRISPR-Cas-Based 
            Recombineering in E. coli through Transient Inhibition of Host RecA 
            Activity.
  JOURNAL   ACS Synth Biol. 2017 Oct 2. doi: 10.1021/acssynbio.7b00174.
  PUBMED    28915012
REFERENCE   2  (bases 1 to 6928)
  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: "ACS Synth 
            Biol. 2017 Oct 2. doi: 10.1021/acssynbio.7b00174."
COMMENT     Sequence Label: pKDsgRNA-pfkB
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    258..276
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        307..382
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             403..432
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             448..465
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(521..538)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      691..737
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(876..1667)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(1668..1759)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(1890..2768)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        2795..3079
                     /gene="araBAD"
                     /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     3002..3021
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             3106..3114
                     /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             3123..3539
                     /codon_start=1
                     /gene="gam"
                     /product="inhibitor of the host RecBCD nuclease in the 
                     lambda Red system"
                     /label=Gam
                     /note="protects the double-stranded DNA substrate for 
                     recombination"
                     /translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY
                     IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR
                     AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV"
     CDS             3545..4330
                     /codon_start=1
                     /gene="bet"
                     /product="single-stranded DNA binding recombinase in the 
                     lambda Red system"
                     /label=Beta
                     /note="mediates pairing of a single-stranded DNA with a 
                     complementary target"
                     /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
                     ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
                     TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
                     FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
                     LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
     CDS             4327..5007
                     /codon_start=1
                     /gene="exo"
                     /product="5' to 3' double-stranded DNA exonuclease in the 
                     lambda Red system"
                     /label=Exo
                     /note="creates 3' single-stranded DNA tails"
                     /translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPR
                     SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
                     IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
                     VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
                     EQWR"
     terminator      5008..5252
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(5375..6325)
                     /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)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      6373..6595
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 ggtctgctat gtggtgctat ctgacttttt gctgttcagc agttcctgcc ctctgatttt
       61 ccagtctgac cacttcggat tatcccgtga caggtcattc agactggcta atgcacccag
      121 taaggcagcg gtatcatcaa cggggtctga cgctcagtgg aacgaaaact cacgttaagg
      181 gattttggtc atgagattat cagctttcgc taaggatgat ttctggaatt cttccctatc
      241 agtgatagag attgacatcc ctatcagtga tagagatact gagcaccgcc ggtcgcgcca
      301 cccgccgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
      361 aaaagtggca ccgagtcggt gctttttttg aagcttgggc ccgaacaaaa actcatctca
      421 gaagaggatc tgaatagcgc cgtcgaccat catcatcatc atcattgagt ttaaacggtc
      481 tccagcttgg ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag
      541 aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac
      601 ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc
      661 cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac
      721 tgggcctttc gttttatctg ttgtttgtcg gtgaactgga tccttactcg agtctagact
      781 gcaggcggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
      841 gtatatatga gtaaacttgg tctgctgaca ggacattatt tgccgactac cttggtgatc
      901 tcgcctttca cgtagtggac aaattcttcc aactgatctg cgcgcgaggc caagcgatct
      961 tcttcttgtc caagataagc ctgtctagct tcaagtatga cgggctgata ctgggccggc
     1021 aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg cgattttgcc ggttactgcg
     1081 ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct catcgccagc ccagtcgggc
     1141 ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa atagatcctg ttcaggaacc
     1201 ggatcaaaga gttcctccgc cgctggacct accaaggcaa cgctatgttc tcttgctttt
     1261 gtcagcaaga tagccagatc aatgtcgatc gtggctggct cgaagatacc tgcaagaatg
     1321 tcattgcgct gccattctcc aaattgcagt tcgcgcttag ctggataacg ccacggaatg
     1381 atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga gaatctcgct ctctccaggg
     1441 gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc gcgttgtttc atcaagcctt
     1501 acggtcaccg taaccagcaa atcaatatca ctgtgtggct tcaggccgcc atccactgcg
     1561 gagccgtaca aatgtacggc cagcaacgtc ggttcgagat ggcgctcgat gacgccaact
     1621 acctctgata gttgagtcga tacttcggcg atcaccgctt ccctcatact cttccttttt
     1681 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     1741 atttagaaaa ataaacaaat agctagctca ctcggtcgct accagggtta ttgtctcatg
     1801 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     1861 ccccgaaaag tgccacctgc atcgatttat tatgacaact tgacggctac atcattcact
     1921 ttttcttcac aaccggcacg gaactcgctc gggctggccc cggtgcattt tttaaatacc
     1981 cgcgagaagt agagttgatc gtcaaaacca acattgcgac cgacggtggc gataggcatc
     2041 cgggtggtgc tcaaaagcag cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag
     2101 acgctaatcc ctaactgctg gcggaaaaga tgtgacagac gcgacggcga caagcaaaca
     2161 tgctgtgcga cgctggcgat atcaaaattg ctgtctgcca ggtgatcgct gatgtactga
     2221 caagcctcgc gtacccgatt atccatcggt ggatggagcg actcgttaat cgcttccatg
     2281 cgccgcagta acaattgctc aagcagattt atcgccagca gctccgaata gcgcccttcc
     2341 ccttgcccgg cgttaatgat ttgcccaaac aggtcgctga aatgcggctg gtgcgcttca
     2401 tccgggcgaa agaaccccgt attggcaaat attgacggcc agttaagcca ttcatgccag
     2461 taggcgcgcg gacgaaagta aacccactgg tgataccatt cgcgagcctc cggatgacga
     2521 ccgtagtgat gaatctctcc tggcgggaac agcaaaatat cacccggtcg gcaaacaaat
     2581 tctcgtccct gatttttcac caccccctga ccgcgaatgg tgagattgag aatataacct
     2641 ttcattccca gcggtcggtc gataaaaaaa tcgagataac cgttggcctc aatcggcgtt
     2701 aaacccgcca ccagatgggc attaaacgag tatcccggca gcaggggatc attttgcgct
     2761 tcagccatac ttttcatact cccgccattc agagaagaaa ccaattgtcc atattgcatc
     2821 agacattgcc gtcactgcgt cttttactgg ctcttctcgc taaccaaacc ggtaaccccg
     2881 cttattaaaa gcattctgta acaaagcggg accaaagcca tgacaaaaac gcgtaacaaa
     2941 agtgtctata atcacggcag aaaagtccac attgattatt tgcacggcgt cacactttgc
     3001 tatgccatag catttttatc cataagatta gcggatccta cctgacgctt tttatcgcaa
     3061 ctctctactg tttctccata cccgtttttt tgggaattcg agctctaagg aggttataaa
     3121 aaatggatat taatactgaa actgagatca agcaaaagca ttcactaacc ccctttcctg
     3181 ttttcctaat cagcccggca tttcgcgggc gatattttca cagctatttc aggagttcag
     3241 ccatgaacgc ttattacatt caggatcgtc ttgaggctca gagctgggcg cgtcactacc
     3301 agcagctcgc ccgtgaagag aaagaggcag aactggcaga cgacatggaa aaaggcctgc
     3361 cccagcacct gtttgaatcg ctatgcatcg atcatttgca acgccacggg gccagcaaaa
     3421 aatccattac ccgtgcgttt gatgacgatg ttgagtttca ggagcgcatg gcagaacaca
     3481 tccggtacat ggttgaaacc attgctcacc accaggttga tattgattca gaggtataaa
     3541 acgaatgagt actgcactcg caacgctggc tgggaagctg gctgaacgtg tcggcatgga
     3601 ttctgtcgac ccacaggaac tgatcaccac tcttcgccag acggcattta aaggtgatgc
     3661 cagcgatgcg cagttcatcg cattactgat cgttgccaac cagtacggcc ttaatccgtg
     3721 gacgaaagaa atttacgcct ttcctgataa gcagaatggc atcgttccgg tggtgggcgt
     3781 tgatggctgg tcccgcatca tcaatgaaaa ccagcagttt gatggcatgg actttgagca
     3841 ggacaatgaa tcctgtacat gccggattta ccgcaaggac cgtaatcatc cgatctgcgt
     3901 taccgaatgg atggatgaat gccgccgcga accattcaaa actcgcgaag gcagagaaat
     3961 cacggggccg tggcagtcgc atcccaaacg gatgttacgt cataaagcca tgattcagtg
     4021 tgcccgtctg gccttcggat ttgctggtat ctatgacaag gatgaagccg agcgcattgt
     4081 cgaaaatact gcatacactg cagaacgtca gccggaacgc gacatcactc cggttaacga
     4141 tgaaaccatg caggagatta acactctgct gatcgccctg gataaaacat gggatgacga
     4201 cttattgccg ctctgttccc agatatttcg ccgcgacatt cgtgcatcgt cagaactgac
     4261 acaggccgaa gcagtaaaag ctcttggatt cctgaaacag aaagccgcag agcagaaggt
     4321 ggcagcatga caccggacat tatcctgcag cgtaccggga tcgatgtgag agctgtcgaa
     4381 cagggggatg atgcgtggca caaattacgg ctcggcgtca tcaccgcttc agaagttcac
     4441 aacgtgatag caaaaccccg ctccggaaag aagtggcctg acatgaaaat gtcctacttc
     4501 cacaccctgc ttgctgaggt ttgcaccggt gtggctccgg aagttaacgc taaagcactg
     4561 gcctggggaa aacagtacga gaacgacgcc agaaccctgt ttgaattcac ttccggcgtg
     4621 aatgttactg aatccccgat catctatcgc gacgaaagta tgcgtaccgc ctgctctccc
     4681 gatggtttat gcagtgacgg caacggcctt gaactgaaat gcccgtttac ctcccgggat
     4741 ttcatgaagt tccggctcgg tggtttcgag gccataaagt cagcttacat ggcccaggtg
     4801 cagtacagca tgtgggtgac gcgaaaaaat gcctggtact ttgccaacta tgacccgcgt
     4861 atgaagcgtg aaggcctgca ttatgtcgtg attgagcggg atgaaaagta catggcgagt
     4921 tttgacgaga tcgtgccgga gttcatcgaa aaaatggacg aggcactggc tgaaattggt
     4981 tttgtatttg gggagcaatg gcgatgacgc atcctcacga taatatccgg gtaggcgcaa
     5041 tcactttcgt ctactccgtt acaaagcgag gctgggtatt tcccggcctt tctgttatcc
     5101 gaaatccact gaaagcacag cggctggctg aggagataaa taataaacga ggggctgtat
     5161 gcacaaagca tcttctgttg agttaagaac gagtatcgag atggcacata gccttgctca
     5221 aattggaatc aggtttgtgc caataccagt agaaacagac gaagaatcca tgggtatgga
     5281 cagttttccc tttgatatgt aacggtgaac agttgttcta cttttgtttg ttagtcttga
     5341 tgcttcactg atagatacaa gagccataag aacctcagat ccttccgtat ttagccagta
     5401 tgttctctag tgtggttcgt tgtttttgcg tgagccatga gaacgaacca ttgagatcat
     5461 acttactttg catgtcactc aaaaattttg cctcaaaact ggtgagctga atttttgcag
     5521 ttaaagcatc gtgtagtgtt tttcttagtc cgttatgtag gtaggaatct gatgtaatgg
     5581 ttgttggtat tttgtcacca ttcattttta tctggttgtt ctcaagttcg gttacgagat
     5641 ccatttgtct atctagttca acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat
     5701 caaccaccaa tttcatattg ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc
     5761 attggttaag ccttttaaac tcatggtagt tattttcaag cattaacatg aacttaaatt
     5821 catcaaggct aatctctata tttgccttgt gagttttctt ttgtgttagt tcttttaata
     5881 accactcata aatcctcata gagtatttgt tttcaaaaga cttaacatgt tccagattat
     5941 attttatgaa tttttttaac tggaaaagat aaggcaatat ctcttcacta aaaactaatt
     6001 ctaatttttc gcttgagaac ttggcatagt ttgtccactg gaaaatctca aagcctttaa
     6061 ccaaaggatt cctgatttcc acagttctcg tcatcagctc tctggttgct ttagctaata
     6121 caccataagc attttcccta ctgatgttca tcatctgaac gtattggtta taagtgaacg
     6181 ataccgtccg ttctttcctt gtagggtttt caatcgtggg gttgagtagt gccacacagc
     6241 ataaaattag cttggtttca tgctccgtta agtcatagcg actaatcgct agttcatttg
     6301 ctttgaaaac aactaattca gacatacatc tcaattggtc taggtgattt taatcactat
     6361 accaattgag atgggctagt caatgataat tactagtcct tttcctttga gttgtgggta
     6421 tctgtaaatt ctgctagacc tttgctggaa aacttgtaaa ttctgctaga ccctctgtaa
     6481 attccgctag acctttgtgt gttttttttg tttatattca agtggttata atttatagaa
     6541 taaagaaaga ataaaaaaag ataaaaagaa tagatcccag ccctgtgtat aactcactac
     6601 tttagtcagt tccgcagtat tacaaaagga tgtcgcaaac gctgtttgct cctctacaaa
     6661 acagacctta aaaccctaaa ggcttaagta gcaccctcgc aagctcggtt gcggccgcaa
     6721 tcgggcaaat cgctgaatat tccttttgtc tccgaccatc aggcacctga gtcgctgtct
     6781 ttttcgtgac attcagttcg ctgcgctcac ggctctggca gtgaatgggg gtaaatggca
     6841 ctacaggcgc cttttatgga ttcatgcaag gaaactaccc ataatacaag aaaagcccgt
     6901 cacgggcttc tcagggcgtt ttatggcg
//
