LOCUS       sequence-181330-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains arabinose-induced lambda Red and a tet-inducible gRNA 
            targeting rpsL..
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-rpsL
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    228..246
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    253..271
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        302..377
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             398..427
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             443..460
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(516..533)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      686..732
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(871..1662)
                     /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(1663..1754)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(1885..2763)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        2790..3074
                     /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     2997..3016
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             3101..3109
                     /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             3118..3534
                     /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             3540..4325
                     /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             4322..5002
                     /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      5003..5247
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(5370..6320)
                     /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      6368..6590
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 gctatgtggt gctatctgac tttttgctgt tcagcagttc ctgccctctg attttccagt
       61 ctgaccactt cggattatcc cgtgacaggt cattcagact ggctaatgca cccagtaagg
      121 cagcggtatc atcaacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
      181 tggtcatgag attatcagct ttcgctaagg atgatttctg gaattcttcc ctatcagtga
      241 tagagattga catccctatc agtgatagag atactgagca cagcgcggag ttcggttttt
      301 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
      361 tggcaccgag tcggtgcttt ttttgaagct tgggcccgaa caaaaactca tctcagaaga
      421 ggatctgaat agcgccgtcg accatcatca tcatcatcat tgagtttaaa cggtctccag
      481 cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa atcagaacgc
      541 agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac
      601 cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat
      661 gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
      721 ctttcgtttt atctgttgtt tgtcggtgaa ctggatcctt actcgagtct agactgcagg
      781 cggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
      841 tatgagtaaa cttggtctgc tgacaggaca ttatttgccg actaccttgg tgatctcgcc
      901 tttcacgtag tggacaaatt cttccaactg atctgcgcgc gaggccaagc gatcttcttc
      961 ttgtccaaga taagcctgtc tagcttcaag tatgacgggc tgatactggg ccggcaggcg
     1021 ctccattgcc cagtcggcag cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta
     1081 ccaaatgcgg gacaacgtaa gcactacatt tcgctcatcg ccagcccagt cgggcggcga
     1141 gttccatagc gttaaggttt catttagcgc ctcaaataga tcctgttcag gaaccggatc
     1201 aaagagttcc tccgccgctg gacctaccaa ggcaacgcta tgttctcttg cttttgtcag
     1261 caagatagcc agatcaatgt cgatcgtggc tggctcgaag atacctgcaa gaatgtcatt
     1321 gcgctgccat tctccaaatt gcagttcgcg cttagctgga taacgccacg gaatgatgtc
     1381 gtcgtgcaca acaatggtga cttctacagc gcggagaatc tcgctctctc caggggaagc
     1441 cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt
     1501 caccgtaacc agcaaatcaa tatcactgtg tggcttcagg ccgccatcca ctgcggagcc
     1561 gtacaaatgt acggccagca acgtcggttc gagatggcgc tcgatgacgc caactacctc
     1621 tgatagttga gtcgatactt cggcgatcac cgcttccctc atactcttcc tttttcaata
     1681 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     1741 gaaaaataaa caaatagcta gctcactcgg tcgctaccag ggttattgtc tcatgagcgg
     1801 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     1861 aaaagtgcca cctgcatcga tttattatga caacttgacg gctacatcat tcactttttc
     1921 ttcacaaccg gcacggaact cgctcgggct ggccccggtg cattttttaa atacccgcga
     1981 gaagtagagt tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag gcatccgggt
     2041 ggtgctcaaa agcagcttcg cctggctgat acgttggtcc tcgcgccagc ttaagacgct
     2101 aatccctaac tgctggcgga aaagatgtga cagacgcgac ggcgacaagc aaacatgctg
     2161 tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt actgacaagc
     2221 ctcgcgtacc cgattatcca tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg
     2281 cagtaacaat tgctcaagca gatttatcgc cagcagctcc gaatagcgcc cttccccttg
     2341 cccggcgtta atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg
     2401 gcgaaagaac cccgtattgg caaatattga cggccagtta agccattcat gccagtaggc
     2461 gcgcggacga aagtaaaccc actggtgata ccattcgcga gcctccggat gacgaccgta
     2521 gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg
     2581 tccctgattt ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat aacctttcat
     2641 tcccagcggt cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc
     2701 cgccaccaga tgggcattaa acgagtatcc cggcagcagg ggatcatttt gcgcttcagc
     2761 catacttttc atactcccgc cattcagaga agaaaccaat tgtccatatt gcatcagaca
     2821 ttgccgtcac tgcgtctttt actggctctt ctcgctaacc aaaccggtaa ccccgcttat
     2881 taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt
     2941 ctataatcac ggcagaaaag tccacattga ttatttgcac ggcgtcacac tttgctatgc
     3001 catagcattt ttatccataa gattagcgga tcctacctga cgctttttat cgcaactctc
     3061 tactgtttct ccatacccgt ttttttggga attcgagctc taaggaggtt ataaaaaatg
     3121 gatattaata ctgaaactga gatcaagcaa aagcattcac taaccccctt tcctgttttc
     3181 ctaatcagcc cggcatttcg cgggcgatat tttcacagct atttcaggag ttcagccatg
     3241 aacgcttatt acattcagga tcgtcttgag gctcagagct gggcgcgtca ctaccagcag
     3301 ctcgcccgtg aagagaaaga ggcagaactg gcagacgaca tggaaaaagg cctgccccag
     3361 cacctgtttg aatcgctatg catcgatcat ttgcaacgcc acggggccag caaaaaatcc
     3421 attacccgtg cgtttgatga cgatgttgag tttcaggagc gcatggcaga acacatccgg
     3481 tacatggttg aaaccattgc tcaccaccag gttgatattg attcagaggt ataaaacgaa
     3541 tgagtactgc actcgcaacg ctggctggga agctggctga acgtgtcggc atggattctg
     3601 tcgacccaca ggaactgatc accactcttc gccagacggc atttaaaggt gatgccagcg
     3661 atgcgcagtt catcgcatta ctgatcgttg ccaaccagta cggccttaat ccgtggacga
     3721 aagaaattta cgcctttcct gataagcaga atggcatcgt tccggtggtg ggcgttgatg
     3781 gctggtcccg catcatcaat gaaaaccagc agtttgatgg catggacttt gagcaggaca
     3841 atgaatcctg tacatgccgg atttaccgca aggaccgtaa tcatccgatc tgcgttaccg
     3901 aatggatgga tgaatgccgc cgcgaaccat tcaaaactcg cgaaggcaga gaaatcacgg
     3961 ggccgtggca gtcgcatccc aaacggatgt tacgtcataa agccatgatt cagtgtgccc
     4021 gtctggcctt cggatttgct ggtatctatg acaaggatga agccgagcgc attgtcgaaa
     4081 atactgcata cactgcagaa cgtcagccgg aacgcgacat cactccggtt aacgatgaaa
     4141 ccatgcagga gattaacact ctgctgatcg ccctggataa aacatgggat gacgacttat
     4201 tgccgctctg ttcccagata tttcgccgcg acattcgtgc atcgtcagaa ctgacacagg
     4261 ccgaagcagt aaaagctctt ggattcctga aacagaaagc cgcagagcag aaggtggcag
     4321 catgacaccg gacattatcc tgcagcgtac cgggatcgat gtgagagctg tcgaacaggg
     4381 ggatgatgcg tggcacaaat tacggctcgg cgtcatcacc gcttcagaag ttcacaacgt
     4441 gatagcaaaa ccccgctccg gaaagaagtg gcctgacatg aaaatgtcct acttccacac
     4501 cctgcttgct gaggtttgca ccggtgtggc tccggaagtt aacgctaaag cactggcctg
     4561 gggaaaacag tacgagaacg acgccagaac cctgtttgaa ttcacttccg gcgtgaatgt
     4621 tactgaatcc ccgatcatct atcgcgacga aagtatgcgt accgcctgct ctcccgatgg
     4681 tttatgcagt gacggcaacg gccttgaact gaaatgcccg tttacctccc gggatttcat
     4741 gaagttccgg ctcggtggtt tcgaggccat aaagtcagct tacatggccc aggtgcagta
     4801 cagcatgtgg gtgacgcgaa aaaatgcctg gtactttgcc aactatgacc cgcgtatgaa
     4861 gcgtgaaggc ctgcattatg tcgtgattga gcgggatgaa aagtacatgg cgagttttga
     4921 cgagatcgtg ccggagttca tcgaaaaaat ggacgaggca ctggctgaaa ttggttttgt
     4981 atttggggag caatggcgat gacgcatcct cacgataata tccgggtagg cgcaatcact
     5041 ttcgtctact ccgttacaaa gcgaggctgg gtatttcccg gcctttctgt tatccgaaat
     5101 ccactgaaag cacagcggct ggctgaggag ataaataata aacgaggggc tgtatgcaca
     5161 aagcatcttc tgttgagtta agaacgagta tcgagatggc acatagcctt gctcaaattg
     5221 gaatcaggtt tgtgccaata ccagtagaaa cagacgaaga atccatgggt atggacagtt
     5281 ttccctttga tatgtaacgg tgaacagttg ttctactttt gtttgttagt cttgatgctt
     5341 cactgataga tacaagagcc ataagaacct cagatccttc cgtatttagc cagtatgttc
     5401 tctagtgtgg ttcgttgttt ttgcgtgagc catgagaacg aaccattgag atcatactta
     5461 ctttgcatgt cactcaaaaa ttttgcctca aaactggtga gctgaatttt tgcagttaaa
     5521 gcatcgtgta gtgtttttct tagtccgtta tgtaggtagg aatctgatgt aatggttgtt
     5581 ggtattttgt caccattcat ttttatctgg ttgttctcaa gttcggttac gagatccatt
     5641 tgtctatcta gttcaacttg gaaaatcaac gtatcagtcg ggcggcctcg cttatcaacc
     5701 accaatttca tattgctgta agtgtttaaa tctttactta ttggtttcaa aacccattgg
     5761 ttaagccttt taaactcatg gtagttattt tcaagcatta acatgaactt aaattcatca
     5821 aggctaatct ctatatttgc cttgtgagtt ttcttttgtg ttagttcttt taataaccac
     5881 tcataaatcc tcatagagta tttgttttca aaagacttaa catgttccag attatatttt
     5941 atgaattttt ttaactggaa aagataaggc aatatctctt cactaaaaac taattctaat
     6001 ttttcgcttg agaacttggc atagtttgtc cactggaaaa tctcaaagcc tttaaccaaa
     6061 ggattcctga tttccacagt tctcgtcatc agctctctgg ttgctttagc taatacacca
     6121 taagcatttt ccctactgat gttcatcatc tgaacgtatt ggttataagt gaacgatacc
     6181 gtccgttctt tccttgtagg gttttcaatc gtggggttga gtagtgccac acagcataaa
     6241 attagcttgg tttcatgctc cgttaagtca tagcgactaa tcgctagttc atttgctttg
     6301 aaaacaacta attcagacat acatctcaat tggtctaggt gattttaatc actataccaa
     6361 ttgagatggg ctagtcaatg ataattacta gtccttttcc tttgagttgt gggtatctgt
     6421 aaattctgct agacctttgc tggaaaactt gtaaattctg ctagaccctc tgtaaattcc
     6481 gctagacctt tgtgtgtttt ttttgtttat attcaagtgg ttataattta tagaataaag
     6541 aaagaataaa aaaagataaa aagaatagat cccagccctg tgtataactc actactttag
     6601 tcagttccgc agtattacaa aaggatgtcg caaacgctgt ttgctcctct acaaaacaga
     6661 ccttaaaacc ctaaaggctt aagtagcacc ctcgcaagct cggttgcggc cgcaatcggg
     6721 caaatcgctg aatattcctt ttgtctccga ccatcaggca cctgagtcgc tgtctttttc
     6781 gtgacattca gttcgctgcg ctcacggctc tggcagtgaa tgggggtaaa tggcactaca
     6841 ggcgcctttt atggattcat gcaaggaaac tacccataat acaagaaaag cccgtcacgg
     6901 gcttctcagg gcgttttatg gcgggtct
//
