LOCUS       sequence-181336-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains arabinose-induced lambda Red and a tet-inducible gRNA 
            targeting yhhT..
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-yhhT
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(9..887)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        914..1198
                     /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     1121..1140
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             1225..1233
                     /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             1242..1658
                     /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             1664..2449
                     /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             2446..3126
                     /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      3127..3371
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(3494..4444)
                     /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      4492..4714
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     protein_bind    5280..5298
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5305..5323
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        5354..5429
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             5450..5479
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5495..5512
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5568..5585)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5738..5784
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5923..6714)
                     /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(6715..6806)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgatttatt atgacaactt gacggctaca tcattcactt tttcttcaca accggcacgg
       61 aactcgctcg ggctggcccc ggtgcatttt ttaaataccc gcgagaagta gagttgatcg
      121 tcaaaaccaa cattgcgacc gacggtggcg ataggcatcc gggtggtgct caaaagcagc
      181 ttcgcctggc tgatacgttg gtcctcgcgc cagcttaaga cgctaatccc taactgctgg
      241 cggaaaagat gtgacagacg cgacggcgac aagcaaacat gctgtgcgac gctggcgata
      301 tcaaaattgc tgtctgccag gtgatcgctg atgtactgac aagcctcgcg tacccgatta
      361 tccatcggtg gatggagcga ctcgttaatc gcttccatgc gccgcagtaa caattgctca
      421 agcagattta tcgccagcag ctccgaatag cgcccttccc cttgcccggc gttaatgatt
      481 tgcccaaaca ggtcgctgaa atgcggctgg tgcgcttcat ccgggcgaaa gaaccccgta
      541 ttggcaaata ttgacggcca gttaagccat tcatgccagt aggcgcgcgg acgaaagtaa
      601 acccactggt gataccattc gcgagcctcc ggatgacgac cgtagtgatg aatctctcct
      661 ggcgggaaca gcaaaatatc acccggtcgg caaacaaatt ctcgtccctg atttttcacc
      721 accccctgac cgcgaatggt gagattgaga atataacctt tcattcccag cggtcggtcg
      781 ataaaaaaat cgagataacc gttggcctca atcggcgtta aacccgccac cagatgggca
      841 ttaaacgagt atcccggcag caggggatca ttttgcgctt cagccatact tttcatactc
      901 ccgccattca gagaagaaac caattgtcca tattgcatca gacattgccg tcactgcgtc
      961 ttttactggc tcttctcgct aaccaaaccg gtaaccccgc ttattaaaag cattctgtaa
     1021 caaagcggga ccaaagccat gacaaaaacg cgtaacaaaa gtgtctataa tcacggcaga
     1081 aaagtccaca ttgattattt gcacggcgtc acactttgct atgccatagc atttttatcc
     1141 ataagattag cggatcctac ctgacgcttt ttatcgcaac tctctactgt ttctccatac
     1201 ccgttttttt gggaattcga gctctaagga ggttataaaa aatggatatt aatactgaaa
     1261 ctgagatcaa gcaaaagcat tcactaaccc cctttcctgt tttcctaatc agcccggcat
     1321 ttcgcgggcg atattttcac agctatttca ggagttcagc catgaacgct tattacattc
     1381 aggatcgtct tgaggctcag agctgggcgc gtcactacca gcagctcgcc cgtgaagaga
     1441 aagaggcaga actggcagac gacatggaaa aaggcctgcc ccagcacctg tttgaatcgc
     1501 tatgcatcga tcatttgcaa cgccacgggg ccagcaaaaa atccattacc cgtgcgtttg
     1561 atgacgatgt tgagtttcag gagcgcatgg cagaacacat ccggtacatg gttgaaacca
     1621 ttgctcacca ccaggttgat attgattcag aggtataaaa cgaatgagta ctgcactcgc
     1681 aacgctggct gggaagctgg ctgaacgtgt cggcatggat tctgtcgacc cacaggaact
     1741 gatcaccact cttcgccaga cggcatttaa aggtgatgcc agcgatgcgc agttcatcgc
     1801 attactgatc gttgccaacc agtacggcct taatccgtgg acgaaagaaa tttacgcctt
     1861 tcctgataag cagaatggca tcgttccggt ggtgggcgtt gatggctggt cccgcatcat
     1921 caatgaaaac cagcagtttg atggcatgga ctttgagcag gacaatgaat cctgtacatg
     1981 ccggatttac cgcaaggacc gtaatcatcc gatctgcgtt accgaatgga tggatgaatg
     2041 ccgccgcgaa ccattcaaaa ctcgcgaagg cagagaaatc acggggccgt ggcagtcgca
     2101 tcccaaacgg atgttacgtc ataaagccat gattcagtgt gcccgtctgg ccttcggatt
     2161 tgctggtatc tatgacaagg atgaagccga gcgcattgtc gaaaatactg catacactgc
     2221 agaacgtcag ccggaacgcg acatcactcc ggttaacgat gaaaccatgc aggagattaa
     2281 cactctgctg atcgccctgg ataaaacatg ggatgacgac ttattgccgc tctgttccca
     2341 gatatttcgc cgcgacattc gtgcatcgtc agaactgaca caggccgaag cagtaaaagc
     2401 tcttggattc ctgaaacaga aagccgcaga gcagaaggtg gcagcatgac accggacatt
     2461 atcctgcagc gtaccgggat cgatgtgaga gctgtcgaac agggggatga tgcgtggcac
     2521 aaattacggc tcggcgtcat caccgcttca gaagttcaca acgtgatagc aaaaccccgc
     2581 tccggaaaga agtggcctga catgaaaatg tcctacttcc acaccctgct tgctgaggtt
     2641 tgcaccggtg tggctccgga agttaacgct aaagcactgg cctggggaaa acagtacgag
     2701 aacgacgcca gaaccctgtt tgaattcact tccggcgtga atgttactga atccccgatc
     2761 atctatcgcg acgaaagtat gcgtaccgcc tgctctcccg atggtttatg cagtgacggc
     2821 aacggccttg aactgaaatg cccgtttacc tcccgggatt tcatgaagtt ccggctcggt
     2881 ggtttcgagg ccataaagtc agcttacatg gcccaggtgc agtacagcat gtgggtgacg
     2941 cgaaaaaatg cctggtactt tgccaactat gacccgcgta tgaagcgtga aggcctgcat
     3001 tatgtcgtga ttgagcggga tgaaaagtac atggcgagtt ttgacgagat cgtgccggag
     3061 ttcatcgaaa aaatggacga ggcactggct gaaattggtt ttgtatttgg ggagcaatgg
     3121 cgatgacgca tcctcacgat aatatccggg taggcgcaat cactttcgtc tactccgtta
     3181 caaagcgagg ctgggtattt cccggccttt ctgttatccg aaatccactg aaagcacagc
     3241 ggctggctga ggagataaat aataaacgag gggctgtatg cacaaagcat cttctgttga
     3301 gttaagaacg agtatcgaga tggcacatag ccttgctcaa attggaatca ggtttgtgcc
     3361 aataccagta gaaacagacg aagaatccat gggtatggac agttttccct ttgatatgta
     3421 acggtgaaca gttgttctac ttttgtttgt tagtcttgat gcttcactga tagatacaag
     3481 agccataaga acctcagatc cttccgtatt tagccagtat gttctctagt gtggttcgtt
     3541 gtttttgcgt gagccatgag aacgaaccat tgagatcata cttactttgc atgtcactca
     3601 aaaattttgc ctcaaaactg gtgagctgaa tttttgcagt taaagcatcg tgtagtgttt
     3661 ttcttagtcc gttatgtagg taggaatctg atgtaatggt tgttggtatt ttgtcaccat
     3721 tcatttttat ctggttgttc tcaagttcgg ttacgagatc catttgtcta tctagttcaa
     3781 cttggaaaat caacgtatca gtcgggcggc ctcgcttatc aaccaccaat ttcatattgc
     3841 tgtaagtgtt taaatcttta cttattggtt tcaaaaccca ttggttaagc cttttaaact
     3901 catggtagtt attttcaagc attaacatga acttaaattc atcaaggcta atctctatat
     3961 ttgccttgtg agttttcttt tgtgttagtt cttttaataa ccactcataa atcctcatag
     4021 agtatttgtt ttcaaaagac ttaacatgtt ccagattata ttttatgaat ttttttaact
     4081 ggaaaagata aggcaatatc tcttcactaa aaactaattc taatttttcg cttgagaact
     4141 tggcatagtt tgtccactgg aaaatctcaa agcctttaac caaaggattc ctgatttcca
     4201 cagttctcgt catcagctct ctggttgctt tagctaatac accataagca ttttccctac
     4261 tgatgttcat catctgaacg tattggttat aagtgaacga taccgtccgt tctttccttg
     4321 tagggttttc aatcgtgggg ttgagtagtg ccacacagca taaaattagc ttggtttcat
     4381 gctccgttaa gtcatagcga ctaatcgcta gttcatttgc tttgaaaaca actaattcag
     4441 acatacatct caattggtct aggtgatttt aatcactata ccaattgaga tgggctagtc
     4501 aatgataatt actagtcctt ttcctttgag ttgtgggtat ctgtaaattc tgctagacct
     4561 ttgctggaaa acttgtaaat tctgctagac cctctgtaaa ttccgctaga cctttgtgtg
     4621 ttttttttgt ttatattcaa gtggttataa tttatagaat aaagaaagaa taaaaaaaga
     4681 taaaaagaat agatcccagc cctgtgtata actcactact ttagtcagtt ccgcagtatt
     4741 acaaaaggat gtcgcaaacg ctgtttgctc ctctacaaaa cagaccttaa aaccctaaag
     4801 gcttaagtag caccctcgca agctcggttg cggccgcaat cgggcaaatc gctgaatatt
     4861 ccttttgtct ccgaccatca ggcacctgag tcgctgtctt tttcgtgaca ttcagttcgc
     4921 tgcgctcacg gctctggcag tgaatggggg taaatggcac tacaggcgcc ttttatggat
     4981 tcatgcaagg aaactaccca taatacaaga aaagcccgtc acgggcttct cagggcgttt
     5041 tatggcgggt ctgctatgtg gtgctatctg actttttgct gttcagcagt tcctgccctc
     5101 tgattttcca gtctgaccac ttcggattat cccgtgacag gtcattcaga ctggctaatg
     5161 cacccagtaa ggcagcggta tcatcaacgg ggtctgacgc tcagtggaac gaaaactcac
     5221 gttaagggat tttggtcatg agattatcag ctttcgctaa ggatgatttc tggaattctt
     5281 ccctatcagt gatagagatt gacatcccta tcagtgatag agatactgag cacttaattt
     5341 ggggatggct gctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     5401 aacttgaaaa agtggcaccg agtcggtgct ttttttgaag cttgggcccg aacaaaaact
     5461 catctcagaa gaggatctga atagcgccgt cgaccatcat catcatcatc attgagttta
     5521 aacggtctcc agcttggctg ttttggcgga tgagagaaga ttttcagcct gatacagatt
     5581 aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg
     5641 gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg
     5701 gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc
     5761 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aactggatcc ttactcgagt
     5821 ctagactgca ggcggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5881 atctaaagta tatatgagta aacttggtct gctgacagga cattatttgc cgactacctt
     5941 ggtgatctcg cctttcacgt agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa
     6001 gcgatcttct tcttgtccaa gataagcctg tctagcttca agtatgacgg gctgatactg
     6061 ggccggcagg cgctccattg cccagtcggc agcgacatcc ttcggcgcga ttttgccggt
     6121 tactgcgctg taccaaatgc gggacaacgt aagcactaca tttcgctcat cgccagccca
     6181 gtcgggcggc gagttccata gcgttaaggt ttcatttagc gcctcaaata gatcctgttc
     6241 aggaaccgga tcaaagagtt cctccgccgc tggacctacc aaggcaacgc tatgttctct
     6301 tgcttttgtc agcaagatag ccagatcaat gtcgatcgtg gctggctcga agatacctgc
     6361 aagaatgtca ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg gataacgcca
     6421 cggaatgatg tcgtcgtgca caacaatggt gacttctaca gcgcggagaa tctcgctctc
     6481 tccaggggaa gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc
     6541 aagccttacg gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca ggccgccatc
     6601 cactgcggag ccgtacaaat gtacggccag caacgtcggt tcgagatggc gctcgatgac
     6661 gccaactacc tctgatagtt gagtcgatac ttcggcgatc accgcttccc tcatactctt
     6721 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     6781 tgaatgtatt tagaaaaata aacaaatagc tagctcactc ggtcgctacc agggttattg
     6841 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6901 cacatttccc cgaaaagtgc cacctgca
//
