LOCUS       sequence-181337-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains arabinose-induced lambda Red and a tet-inducible gRNA 
            targeting lpoB..
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-lpoB
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(4..882)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        909..1193
                     /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     1116..1135
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             1220..1228
                     /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             1237..1653
                     /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             1659..2444
                     /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             2441..3121
                     /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      3122..3366
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(3489..4439)
                     /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      4487..4709
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     protein_bind    5275..5293
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5300..5318
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        5349..5424
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             5445..5474
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5490..5507
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5563..5580)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5733..5779
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5918..6709)
                     /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(6710..6801)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttattatgac aacttgacgg ctacatcatt cactttttct tcacaaccgg cacggaactc
       61 gctcgggctg gccccggtgc attttttaaa tacccgcgag aagtagagtt gatcgtcaaa
      121 accaacattg cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc
      181 ctggctgata cgttggtcct cgcgccagct taagacgcta atccctaact gctggcggaa
      241 aagatgtgac agacgcgacg gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa
      301 attgctgtct gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc gattatccat
      361 cggtggatgg agcgactcgt taatcgcttc catgcgccgc agtaacaatt gctcaagcag
      421 atttatcgcc agcagctccg aatagcgccc ttccccttgc ccggcgttaa tgatttgccc
      481 aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc
      541 aaatattgac ggccagttaa gccattcatg ccagtaggcg cgcggacgaa agtaaaccca
      601 ctggtgatac cattcgcgag cctccggatg acgaccgtag tgatgaatct ctcctggcgg
      661 gaacagcaaa atatcacccg gtcggcaaac aaattctcgt ccctgatttt tcaccacccc
      721 ctgaccgcga atggtgagat tgagaatata acctttcatt cccagcggtc ggtcgataaa
      781 aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc gccaccagat gggcattaaa
      841 cgagtatccc ggcagcaggg gatcattttg cgcttcagcc atacttttca tactcccgcc
      901 attcagagaa gaaaccaatt gtccatattg catcagacat tgccgtcact gcgtctttta
      961 ctggctcttc tcgctaacca aaccggtaac cccgcttatt aaaagcattc tgtaacaaag
     1021 cgggaccaaa gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt
     1081 ccacattgat tatttgcacg gcgtcacact ttgctatgcc atagcatttt tatccataag
     1141 attagcggat cctacctgac gctttttatc gcaactctct actgtttctc catacccgtt
     1201 tttttgggaa ttcgagctct aaggaggtta taaaaaatgg atattaatac tgaaactgag
     1261 atcaagcaaa agcattcact aacccccttt cctgttttcc taatcagccc ggcatttcgc
     1321 gggcgatatt ttcacagcta tttcaggagt tcagccatga acgcttatta cattcaggat
     1381 cgtcttgagg ctcagagctg ggcgcgtcac taccagcagc tcgcccgtga agagaaagag
     1441 gcagaactgg cagacgacat ggaaaaaggc ctgccccagc acctgtttga atcgctatgc
     1501 atcgatcatt tgcaacgcca cggggccagc aaaaaatcca ttacccgtgc gtttgatgac
     1561 gatgttgagt ttcaggagcg catggcagaa cacatccggt acatggttga aaccattgct
     1621 caccaccagg ttgatattga ttcagaggta taaaacgaat gagtactgca ctcgcaacgc
     1681 tggctgggaa gctggctgaa cgtgtcggca tggattctgt cgacccacag gaactgatca
     1741 ccactcttcg ccagacggca tttaaaggtg atgccagcga tgcgcagttc atcgcattac
     1801 tgatcgttgc caaccagtac ggccttaatc cgtggacgaa agaaatttac gcctttcctg
     1861 ataagcagaa tggcatcgtt ccggtggtgg gcgttgatgg ctggtcccgc atcatcaatg
     1921 aaaaccagca gtttgatggc atggactttg agcaggacaa tgaatcctgt acatgccgga
     1981 tttaccgcaa ggaccgtaat catccgatct gcgttaccga atggatggat gaatgccgcc
     2041 gcgaaccatt caaaactcgc gaaggcagag aaatcacggg gccgtggcag tcgcatccca
     2101 aacggatgtt acgtcataaa gccatgattc agtgtgcccg tctggccttc ggatttgctg
     2161 gtatctatga caaggatgaa gccgagcgca ttgtcgaaaa tactgcatac actgcagaac
     2221 gtcagccgga acgcgacatc actccggtta acgatgaaac catgcaggag attaacactc
     2281 tgctgatcgc cctggataaa acatgggatg acgacttatt gccgctctgt tcccagatat
     2341 ttcgccgcga cattcgtgca tcgtcagaac tgacacaggc cgaagcagta aaagctcttg
     2401 gattcctgaa acagaaagcc gcagagcaga aggtggcagc atgacaccgg acattatcct
     2461 gcagcgtacc gggatcgatg tgagagctgt cgaacagggg gatgatgcgt ggcacaaatt
     2521 acggctcggc gtcatcaccg cttcagaagt tcacaacgtg atagcaaaac cccgctccgg
     2581 aaagaagtgg cctgacatga aaatgtccta cttccacacc ctgcttgctg aggtttgcac
     2641 cggtgtggct ccggaagtta acgctaaagc actggcctgg ggaaaacagt acgagaacga
     2701 cgccagaacc ctgtttgaat tcacttccgg cgtgaatgtt actgaatccc cgatcatcta
     2761 tcgcgacgaa agtatgcgta ccgcctgctc tcccgatggt ttatgcagtg acggcaacgg
     2821 ccttgaactg aaatgcccgt ttacctcccg ggatttcatg aagttccggc tcggtggttt
     2881 cgaggccata aagtcagctt acatggccca ggtgcagtac agcatgtggg tgacgcgaaa
     2941 aaatgcctgg tactttgcca actatgaccc gcgtatgaag cgtgaaggcc tgcattatgt
     3001 cgtgattgag cgggatgaaa agtacatggc gagttttgac gagatcgtgc cggagttcat
     3061 cgaaaaaatg gacgaggcac tggctgaaat tggttttgta tttggggagc aatggcgatg
     3121 acgcatcctc acgataatat ccgggtaggc gcaatcactt tcgtctactc cgttacaaag
     3181 cgaggctggg tatttcccgg cctttctgtt atccgaaatc cactgaaagc acagcggctg
     3241 gctgaggaga taaataataa acgaggggct gtatgcacaa agcatcttct gttgagttaa
     3301 gaacgagtat cgagatggca catagccttg ctcaaattgg aatcaggttt gtgccaatac
     3361 cagtagaaac agacgaagaa tccatgggta tggacagttt tccctttgat atgtaacggt
     3421 gaacagttgt tctacttttg tttgttagtc ttgatgcttc actgatagat acaagagcca
     3481 taagaacctc agatccttcc gtatttagcc agtatgttct ctagtgtggt tcgttgtttt
     3541 tgcgtgagcc atgagaacga accattgaga tcatacttac tttgcatgtc actcaaaaat
     3601 tttgcctcaa aactggtgag ctgaattttt gcagttaaag catcgtgtag tgtttttctt
     3661 agtccgttat gtaggtagga atctgatgta atggttgttg gtattttgtc accattcatt
     3721 tttatctggt tgttctcaag ttcggttacg agatccattt gtctatctag ttcaacttgg
     3781 aaaatcaacg tatcagtcgg gcggcctcgc ttatcaacca ccaatttcat attgctgtaa
     3841 gtgtttaaat ctttacttat tggtttcaaa acccattggt taagcctttt aaactcatgg
     3901 tagttatttt caagcattaa catgaactta aattcatcaa ggctaatctc tatatttgcc
     3961 ttgtgagttt tcttttgtgt tagttctttt aataaccact cataaatcct catagagtat
     4021 ttgttttcaa aagacttaac atgttccaga ttatatttta tgaatttttt taactggaaa
     4081 agataaggca atatctcttc actaaaaact aattctaatt tttcgcttga gaacttggca
     4141 tagtttgtcc actggaaaat ctcaaagcct ttaaccaaag gattcctgat ttccacagtt
     4201 ctcgtcatca gctctctggt tgctttagct aatacaccat aagcattttc cctactgatg
     4261 ttcatcatct gaacgtattg gttataagtg aacgataccg tccgttcttt ccttgtaggg
     4321 ttttcaatcg tggggttgag tagtgccaca cagcataaaa ttagcttggt ttcatgctcc
     4381 gttaagtcat agcgactaat cgctagttca tttgctttga aaacaactaa ttcagacata
     4441 catctcaatt ggtctaggtg attttaatca ctataccaat tgagatgggc tagtcaatga
     4501 taattactag tccttttcct ttgagttgtg ggtatctgta aattctgcta gacctttgct
     4561 ggaaaacttg taaattctgc tagaccctct gtaaattccg ctagaccttt gtgtgttttt
     4621 tttgtttata ttcaagtggt tataatttat agaataaaga aagaataaaa aaagataaaa
     4681 agaatagatc ccagccctgt gtataactca ctactttagt cagttccgca gtattacaaa
     4741 aggatgtcgc aaacgctgtt tgctcctcta caaaacagac cttaaaaccc taaaggctta
     4801 agtagcaccc tcgcaagctc ggttgcggcc gcaatcgggc aaatcgctga atattccttt
     4861 tgtctccgac catcaggcac ctgagtcgct gtctttttcg tgacattcag ttcgctgcgc
     4921 tcacggctct ggcagtgaat gggggtaaat ggcactacag gcgcctttta tggattcatg
     4981 caaggaaact acccataata caagaaaagc ccgtcacggg cttctcaggg cgttttatgg
     5041 cgggtctgct atgtggtgct atctgacttt ttgctgttca gcagttcctg ccctctgatt
     5101 ttccagtctg accacttcgg attatcccgt gacaggtcat tcagactggc taatgcaccc
     5161 agtaaggcag cggtatcatc aacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5221 gggattttgg tcatgagatt atcagctttc gctaaggatg atttctggaa ttcttcccta
     5281 tcagtgatag agattgacat ccctatcagt gatagagata ctgagcacca gcgcatttcg
     5341 cagcgtttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     5401 gaaaaagtgg caccgagtcg gtgctttttt tgaagcttgg gcccgaacaa aaactcatct
     5461 cagaagagga tctgaatagc gccgtcgacc atcatcatca tcatcattga gtttaaacgg
     5521 tctccagctt ggctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc
     5581 agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc
     5641 acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc
     5701 tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag
     5761 actgggcctt tcgttttatc tgttgtttgt cggtgaactg gatccttact cgagtctaga
     5821 ctgcaggcgg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5881 aagtatatat gagtaaactt ggtctgctga caggacatta tttgccgact accttggtga
     5941 tctcgccttt cacgtagtgg acaaattctt ccaactgatc tgcgcgcgag gccaagcgat
     6001 cttcttcttg tccaagataa gcctgtctag cttcaagtat gacgggctga tactgggccg
     6061 gcaggcgctc cattgcccag tcggcagcga catccttcgg cgcgattttg ccggttactg
     6121 cgctgtacca aatgcgggac aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg
     6181 gcggcgagtt ccatagcgtt aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa
     6241 ccggatcaaa gagttcctcc gccgctggac ctaccaaggc aacgctatgt tctcttgctt
     6301 ttgtcagcaa gatagccaga tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa
     6361 tgtcattgcg ctgccattct ccaaattgca gttcgcgctt agctggataa cgccacggaa
     6421 tgatgtcgtc gtgcacaaca atggtgactt ctacagcgcg gagaatctcg ctctctccag
     6481 gggaagccga agtttccaaa aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc
     6541 ttacggtcac cgtaaccagc aaatcaatat cactgtgtgg cttcaggccg ccatccactg
     6601 cggagccgta caaatgtacg gccagcaacg tcggttcgag atggcgctcg atgacgccaa
     6661 ctacctctga tagttgagtc gatacttcgg cgatcaccgc ttccctcata ctcttccttt
     6721 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6781 gtatttagaa aaataaacaa atagctagct cactcggtcg ctaccagggt tattgtctca
     6841 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6901 ttccccgaaa agtgccacct gcatcgat
//
