LOCUS       sequence-181344-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains arabinose-induced lambda Red and a tet-inducible gRNA 
            targeting srlA..
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-srlA
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(10..888)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        915..1199
                     /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     1122..1141
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             1226..1234
                     /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             1243..1659
                     /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             1665..2450
                     /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             2447..3127
                     /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      3128..3372
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(3495..4445)
                     /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      4493..4715
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     protein_bind    5281..5299
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5306..5324
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        5355..5430
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             5451..5480
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5496..5513
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5569..5586)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5739..5785
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5924..6715)
                     /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(6716..6807)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atcgatttat tatgacaact tgacggctac atcattcact ttttcttcac aaccggcacg
       61 gaactcgctc gggctggccc cggtgcattt tttaaatacc cgcgagaagt agagttgatc
      121 gtcaaaacca acattgcgac cgacggtggc gataggcatc cgggtggtgc tcaaaagcag
      181 cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg
      241 gcggaaaaga tgtgacagac gcgacggcga caagcaaaca tgctgtgcga cgctggcgat
      301 atcaaaattg ctgtctgcca ggtgatcgct gatgtactga caagcctcgc gtacccgatt
      361 atccatcggt ggatggagcg actcgttaat cgcttccatg cgccgcagta acaattgctc
      421 aagcagattt atcgccagca gctccgaata gcgcccttcc ccttgcccgg cgttaatgat
      481 ttgcccaaac aggtcgctga aatgcggctg gtgcgcttca tccgggcgaa agaaccccgt
      541 attggcaaat attgacggcc agttaagcca ttcatgccag taggcgcgcg gacgaaagta
      601 aacccactgg tgataccatt cgcgagcctc cggatgacga ccgtagtgat gaatctctcc
      661 tggcgggaac agcaaaatat cacccggtcg gcaaacaaat tctcgtccct gatttttcac
      721 caccccctga ccgcgaatgg tgagattgag aatataacct ttcattccca gcggtcggtc
      781 gataaaaaaa tcgagataac cgttggcctc aatcggcgtt aaacccgcca ccagatgggc
      841 attaaacgag tatcccggca gcaggggatc attttgcgct tcagccatac ttttcatact
      901 cccgccattc agagaagaaa ccaattgtcc atattgcatc agacattgcc gtcactgcgt
      961 cttttactgg ctcttctcgc taaccaaacc ggtaaccccg cttattaaaa gcattctgta
     1021 acaaagcggg accaaagcca tgacaaaaac gcgtaacaaa agtgtctata atcacggcag
     1081 aaaagtccac attgattatt tgcacggcgt cacactttgc tatgccatag catttttatc
     1141 cataagatta gcggatccta cctgacgctt tttatcgcaa ctctctactg tttctccata
     1201 cccgtttttt tgggaattcg agctctaagg aggttataaa aaatggatat taatactgaa
     1261 actgagatca agcaaaagca ttcactaacc ccctttcctg ttttcctaat cagcccggca
     1321 tttcgcgggc gatattttca cagctatttc aggagttcag ccatgaacgc ttattacatt
     1381 caggatcgtc ttgaggctca gagctgggcg cgtcactacc agcagctcgc ccgtgaagag
     1441 aaagaggcag aactggcaga cgacatggaa aaaggcctgc cccagcacct gtttgaatcg
     1501 ctatgcatcg atcatttgca acgccacggg gccagcaaaa aatccattac ccgtgcgttt
     1561 gatgacgatg ttgagtttca ggagcgcatg gcagaacaca tccggtacat ggttgaaacc
     1621 attgctcacc accaggttga tattgattca gaggtataaa acgaatgagt actgcactcg
     1681 caacgctggc tgggaagctg gctgaacgtg tcggcatgga ttctgtcgac ccacaggaac
     1741 tgatcaccac tcttcgccag acggcattta aaggtgatgc cagcgatgcg cagttcatcg
     1801 cattactgat cgttgccaac cagtacggcc ttaatccgtg gacgaaagaa atttacgcct
     1861 ttcctgataa gcagaatggc atcgttccgg tggtgggcgt tgatggctgg tcccgcatca
     1921 tcaatgaaaa ccagcagttt gatggcatgg actttgagca ggacaatgaa tcctgtacat
     1981 gccggattta ccgcaaggac cgtaatcatc cgatctgcgt taccgaatgg atggatgaat
     2041 gccgccgcga accattcaaa actcgcgaag gcagagaaat cacggggccg tggcagtcgc
     2101 atcccaaacg gatgttacgt cataaagcca tgattcagtg tgcccgtctg gccttcggat
     2161 ttgctggtat ctatgacaag gatgaagccg agcgcattgt cgaaaatact gcatacactg
     2221 cagaacgtca gccggaacgc gacatcactc cggttaacga tgaaaccatg caggagatta
     2281 acactctgct gatcgccctg gataaaacat gggatgacga cttattgccg ctctgttccc
     2341 agatatttcg ccgcgacatt cgtgcatcgt cagaactgac acaggccgaa gcagtaaaag
     2401 ctcttggatt cctgaaacag aaagccgcag agcagaaggt ggcagcatga caccggacat
     2461 tatcctgcag cgtaccggga tcgatgtgag agctgtcgaa cagggggatg atgcgtggca
     2521 caaattacgg ctcggcgtca tcaccgcttc agaagttcac aacgtgatag caaaaccccg
     2581 ctccggaaag aagtggcctg acatgaaaat gtcctacttc cacaccctgc ttgctgaggt
     2641 ttgcaccggt gtggctccgg aagttaacgc taaagcactg gcctggggaa aacagtacga
     2701 gaacgacgcc agaaccctgt ttgaattcac ttccggcgtg aatgttactg aatccccgat
     2761 catctatcgc gacgaaagta tgcgtaccgc ctgctctccc gatggtttat gcagtgacgg
     2821 caacggcctt gaactgaaat gcccgtttac ctcccgggat ttcatgaagt tccggctcgg
     2881 tggtttcgag gccataaagt cagcttacat ggcccaggtg cagtacagca tgtgggtgac
     2941 gcgaaaaaat gcctggtact ttgccaacta tgacccgcgt atgaagcgtg aaggcctgca
     3001 ttatgtcgtg attgagcggg atgaaaagta catggcgagt tttgacgaga tcgtgccgga
     3061 gttcatcgaa aaaatggacg aggcactggc tgaaattggt tttgtatttg gggagcaatg
     3121 gcgatgacgc atcctcacga taatatccgg gtaggcgcaa tcactttcgt ctactccgtt
     3181 acaaagcgag gctgggtatt tcccggcctt tctgttatcc gaaatccact gaaagcacag
     3241 cggctggctg aggagataaa taataaacga ggggctgtat gcacaaagca tcttctgttg
     3301 agttaagaac gagtatcgag atggcacata gccttgctca aattggaatc aggtttgtgc
     3361 caataccagt agaaacagac gaagaatcca tgggtatgga cagttttccc tttgatatgt
     3421 aacggtgaac agttgttcta cttttgtttg ttagtcttga tgcttcactg atagatacaa
     3481 gagccataag aacctcagat ccttccgtat ttagccagta tgttctctag tgtggttcgt
     3541 tgtttttgcg tgagccatga gaacgaacca ttgagatcat acttactttg catgtcactc
     3601 aaaaattttg cctcaaaact ggtgagctga atttttgcag ttaaagcatc gtgtagtgtt
     3661 tttcttagtc cgttatgtag gtaggaatct gatgtaatgg ttgttggtat tttgtcacca
     3721 ttcattttta tctggttgtt ctcaagttcg gttacgagat ccatttgtct atctagttca
     3781 acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat caaccaccaa tttcatattg
     3841 ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc attggttaag ccttttaaac
     3901 tcatggtagt tattttcaag cattaacatg aacttaaatt catcaaggct aatctctata
     3961 tttgccttgt gagttttctt ttgtgttagt tcttttaata accactcata aatcctcata
     4021 gagtatttgt tttcaaaaga cttaacatgt tccagattat attttatgaa tttttttaac
     4081 tggaaaagat aaggcaatat ctcttcacta aaaactaatt ctaatttttc gcttgagaac
     4141 ttggcatagt ttgtccactg gaaaatctca aagcctttaa ccaaaggatt cctgatttcc
     4201 acagttctcg tcatcagctc tctggttgct ttagctaata caccataagc attttcccta
     4261 ctgatgttca tcatctgaac gtattggtta taagtgaacg ataccgtccg ttctttcctt
     4321 gtagggtttt caatcgtggg gttgagtagt gccacacagc ataaaattag cttggtttca
     4381 tgctccgtta agtcatagcg actaatcgct agttcatttg ctttgaaaac aactaattca
     4441 gacatacatc tcaattggtc taggtgattt taatcactat accaattgag atgggctagt
     4501 caatgataat tactagtcct tttcctttga gttgtgggta tctgtaaatt ctgctagacc
     4561 tttgctggaa aacttgtaaa ttctgctaga ccctctgtaa attccgctag acctttgtgt
     4621 gttttttttg tttatattca agtggttata atttatagaa taaagaaaga ataaaaaaag
     4681 ataaaaagaa tagatcccag ccctgtgtat aactcactac tttagtcagt tccgcagtat
     4741 tacaaaagga tgtcgcaaac gctgtttgct cctctacaaa acagacctta aaaccctaaa
     4801 ggcttaagta gcaccctcgc aagctcggtt gcggccgcaa tcgggcaaat cgctgaatat
     4861 tccttttgtc tccgaccatc aggcacctga gtcgctgtct ttttcgtgac attcagttcg
     4921 ctgcgctcac ggctctggca gtgaatgggg gtaaatggca ctacaggcgc cttttatgga
     4981 ttcatgcaag gaaactaccc ataatacaag aaaagcccgt cacgggcttc tcagggcgtt
     5041 ttatggcggg tctgctatgt ggtgctatct gactttttgc tgttcagcag ttcctgccct
     5101 ctgattttcc agtctgacca cttcggatta tcccgtgaca ggtcattcag actggctaat
     5161 gcacccagta aggcagcggt atcatcaacg gggtctgacg ctcagtggaa cgaaaactca
     5221 cgttaaggga ttttggtcat gagattatca gctttcgcta aggatgattt ctggaattct
     5281 tccctatcag tgatagagat tgacatccct atcagtgata gagatactga gcaccaatcc
     5341 gatgacccta agccgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     5401 caacttgaaa aagtggcacc gagtcggtgc tttttttgaa gcttgggccc gaacaaaaac
     5461 tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat cattgagttt
     5521 aaacggtctc cagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat
     5581 taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt
     5641 ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt
     5701 ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt
     5761 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaactggatc cttactcgag
     5821 tctagactgc aggcggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5881 aatctaaagt atatatgagt aaacttggtc tgctgacagg acattatttg ccgactacct
     5941 tggtgatctc gcctttcacg tagtggacaa attcttccaa ctgatctgcg cgcgaggcca
     6001 agcgatcttc ttcttgtcca agataagcct gtctagcttc aagtatgacg ggctgatact
     6061 gggccggcag gcgctccatt gcccagtcgg cagcgacatc cttcggcgcg attttgccgg
     6121 ttactgcgct gtaccaaatg cgggacaacg taagcactac atttcgctca tcgccagccc
     6181 agtcgggcgg cgagttccat agcgttaagg tttcatttag cgcctcaaat agatcctgtt
     6241 caggaaccgg atcaaagagt tcctccgccg ctggacctac caaggcaacg ctatgttctc
     6301 ttgcttttgt cagcaagata gccagatcaa tgtcgatcgt ggctggctcg aagatacctg
     6361 caagaatgtc attgcgctgc cattctccaa attgcagttc gcgcttagct ggataacgcc
     6421 acggaatgat gtcgtcgtgc acaacaatgg tgacttctac agcgcggaga atctcgctct
     6481 ctccagggga agccgaagtt tccaaaaggt cgttgatcaa agctcgccgc gttgtttcat
     6541 caagccttac ggtcaccgta accagcaaat caatatcact gtgtggcttc aggccgccat
     6601 ccactgcgga gccgtacaaa tgtacggcca gcaacgtcgg ttcgagatgg cgctcgatga
     6661 cgccaactac ctctgatagt tgagtcgata cttcggcgat caccgcttcc ctcatactct
     6721 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6781 ttgaatgtat ttagaaaaat aaacaaatag ctagctcact cggtcgctac cagggttatt
     6841 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6901 gcacatttcc ccgaaaagtg ccacctgc
//
