LOCUS       sequence-181342-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains arabinose-induced lambda Red and a tet-inducible gRNA 
            targeting rbsK..
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-rbsK
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(13..891)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        918..1202
                     /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     1125..1144
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             1229..1237
                     /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             1246..1662
                     /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             1668..2453
                     /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             2450..3130
                     /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      3131..3375
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(3498..4448)
                     /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      4496..4718
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     protein_bind    5284..5302
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5309..5327
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        5358..5433
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             5454..5483
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5499..5516
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5572..5589)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5742..5788
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5927..6718)
                     /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(6719..6810)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgcatcgatt tattatgaca acttgacggc tacatcattc actttttctt cacaaccggc
       61 acggaactcg ctcgggctgg ccccggtgca ttttttaaat acccgcgaga agtagagttg
      121 atcgtcaaaa ccaacattgc gaccgacggt ggcgataggc atccgggtgg tgctcaaaag
      181 cagcttcgcc tggctgatac gttggtcctc gcgccagctt aagacgctaa tccctaactg
      241 ctggcggaaa agatgtgaca gacgcgacgg cgacaagcaa acatgctgtg cgacgctggc
      301 gatatcaaaa ttgctgtctg ccaggtgatc gctgatgtac tgacaagcct cgcgtacccg
      361 attatccatc ggtggatgga gcgactcgtt aatcgcttcc atgcgccgca gtaacaattg
      421 ctcaagcaga tttatcgcca gcagctccga atagcgccct tccccttgcc cggcgttaat
      481 gatttgccca aacaggtcgc tgaaatgcgg ctggtgcgct tcatccgggc gaaagaaccc
      541 cgtattggca aatattgacg gccagttaag ccattcatgc cagtaggcgc gcggacgaaa
      601 gtaaacccac tggtgatacc attcgcgagc ctccggatga cgaccgtagt gatgaatctc
      661 tcctggcggg aacagcaaaa tatcacccgg tcggcaaaca aattctcgtc cctgattttt
      721 caccaccccc tgaccgcgaa tggtgagatt gagaatataa cctttcattc ccagcggtcg
      781 gtcgataaaa aaatcgagat aaccgttggc ctcaatcggc gttaaacccg ccaccagatg
      841 ggcattaaac gagtatcccg gcagcagggg atcattttgc gcttcagcca tacttttcat
      901 actcccgcca ttcagagaag aaaccaattg tccatattgc atcagacatt gccgtcactg
      961 cgtcttttac tggctcttct cgctaaccaa accggtaacc ccgcttatta aaagcattct
     1021 gtaacaaagc gggaccaaag ccatgacaaa aacgcgtaac aaaagtgtct ataatcacgg
     1081 cagaaaagtc cacattgatt atttgcacgg cgtcacactt tgctatgcca tagcattttt
     1141 atccataaga ttagcggatc ctacctgacg ctttttatcg caactctcta ctgtttctcc
     1201 atacccgttt ttttgggaat tcgagctcta aggaggttat aaaaaatgga tattaatact
     1261 gaaactgaga tcaagcaaaa gcattcacta accccctttc ctgttttcct aatcagcccg
     1321 gcatttcgcg ggcgatattt tcacagctat ttcaggagtt cagccatgaa cgcttattac
     1381 attcaggatc gtcttgaggc tcagagctgg gcgcgtcact accagcagct cgcccgtgaa
     1441 gagaaagagg cagaactggc agacgacatg gaaaaaggcc tgccccagca cctgtttgaa
     1501 tcgctatgca tcgatcattt gcaacgccac ggggccagca aaaaatccat tacccgtgcg
     1561 tttgatgacg atgttgagtt tcaggagcgc atggcagaac acatccggta catggttgaa
     1621 accattgctc accaccaggt tgatattgat tcagaggtat aaaacgaatg agtactgcac
     1681 tcgcaacgct ggctgggaag ctggctgaac gtgtcggcat ggattctgtc gacccacagg
     1741 aactgatcac cactcttcgc cagacggcat ttaaaggtga tgccagcgat gcgcagttca
     1801 tcgcattact gatcgttgcc aaccagtacg gccttaatcc gtggacgaaa gaaatttacg
     1861 cctttcctga taagcagaat ggcatcgttc cggtggtggg cgttgatggc tggtcccgca
     1921 tcatcaatga aaaccagcag tttgatggca tggactttga gcaggacaat gaatcctgta
     1981 catgccggat ttaccgcaag gaccgtaatc atccgatctg cgttaccgaa tggatggatg
     2041 aatgccgccg cgaaccattc aaaactcgcg aaggcagaga aatcacgggg ccgtggcagt
     2101 cgcatcccaa acggatgtta cgtcataaag ccatgattca gtgtgcccgt ctggccttcg
     2161 gatttgctgg tatctatgac aaggatgaag ccgagcgcat tgtcgaaaat actgcataca
     2221 ctgcagaacg tcagccggaa cgcgacatca ctccggttaa cgatgaaacc atgcaggaga
     2281 ttaacactct gctgatcgcc ctggataaaa catgggatga cgacttattg ccgctctgtt
     2341 cccagatatt tcgccgcgac attcgtgcat cgtcagaact gacacaggcc gaagcagtaa
     2401 aagctcttgg attcctgaaa cagaaagccg cagagcagaa ggtggcagca tgacaccgga
     2461 cattatcctg cagcgtaccg ggatcgatgt gagagctgtc gaacaggggg atgatgcgtg
     2521 gcacaaatta cggctcggcg tcatcaccgc ttcagaagtt cacaacgtga tagcaaaacc
     2581 ccgctccgga aagaagtggc ctgacatgaa aatgtcctac ttccacaccc tgcttgctga
     2641 ggtttgcacc ggtgtggctc cggaagttaa cgctaaagca ctggcctggg gaaaacagta
     2701 cgagaacgac gccagaaccc tgtttgaatt cacttccggc gtgaatgtta ctgaatcccc
     2761 gatcatctat cgcgacgaaa gtatgcgtac cgcctgctct cccgatggtt tatgcagtga
     2821 cggcaacggc cttgaactga aatgcccgtt tacctcccgg gatttcatga agttccggct
     2881 cggtggtttc gaggccataa agtcagctta catggcccag gtgcagtaca gcatgtgggt
     2941 gacgcgaaaa aatgcctggt actttgccaa ctatgacccg cgtatgaagc gtgaaggcct
     3001 gcattatgtc gtgattgagc gggatgaaaa gtacatggcg agttttgacg agatcgtgcc
     3061 ggagttcatc gaaaaaatgg acgaggcact ggctgaaatt ggttttgtat ttggggagca
     3121 atggcgatga cgcatcctca cgataatatc cgggtaggcg caatcacttt cgtctactcc
     3181 gttacaaagc gaggctgggt atttcccggc ctttctgtta tccgaaatcc actgaaagca
     3241 cagcggctgg ctgaggagat aaataataaa cgaggggctg tatgcacaaa gcatcttctg
     3301 ttgagttaag aacgagtatc gagatggcac atagccttgc tcaaattgga atcaggtttg
     3361 tgccaatacc agtagaaaca gacgaagaat ccatgggtat ggacagtttt ccctttgata
     3421 tgtaacggtg aacagttgtt ctacttttgt ttgttagtct tgatgcttca ctgatagata
     3481 caagagccat aagaacctca gatccttccg tatttagcca gtatgttctc tagtgtggtt
     3541 cgttgttttt gcgtgagcca tgagaacgaa ccattgagat catacttact ttgcatgtca
     3601 ctcaaaaatt ttgcctcaaa actggtgagc tgaatttttg cagttaaagc atcgtgtagt
     3661 gtttttctta gtccgttatg taggtaggaa tctgatgtaa tggttgttgg tattttgtca
     3721 ccattcattt ttatctggtt gttctcaagt tcggttacga gatccatttg tctatctagt
     3781 tcaacttgga aaatcaacgt atcagtcggg cggcctcgct tatcaaccac caatttcata
     3841 ttgctgtaag tgtttaaatc tttacttatt ggtttcaaaa cccattggtt aagcctttta
     3901 aactcatggt agttattttc aagcattaac atgaacttaa attcatcaag gctaatctct
     3961 atatttgcct tgtgagtttt cttttgtgtt agttctttta ataaccactc ataaatcctc
     4021 atagagtatt tgttttcaaa agacttaaca tgttccagat tatattttat gaattttttt
     4081 aactggaaaa gataaggcaa tatctcttca ctaaaaacta attctaattt ttcgcttgag
     4141 aacttggcat agtttgtcca ctggaaaatc tcaaagcctt taaccaaagg attcctgatt
     4201 tccacagttc tcgtcatcag ctctctggtt gctttagcta atacaccata agcattttcc
     4261 ctactgatgt tcatcatctg aacgtattgg ttataagtga acgataccgt ccgttctttc
     4321 cttgtagggt tttcaatcgt ggggttgagt agtgccacac agcataaaat tagcttggtt
     4381 tcatgctccg ttaagtcata gcgactaatc gctagttcat ttgctttgaa aacaactaat
     4441 tcagacatac atctcaattg gtctaggtga ttttaatcac tataccaatt gagatgggct
     4501 agtcaatgat aattactagt ccttttcctt tgagttgtgg gtatctgtaa attctgctag
     4561 acctttgctg gaaaacttgt aaattctgct agaccctctg taaattccgc tagacctttg
     4621 tgtgtttttt ttgtttatat tcaagtggtt ataatttata gaataaagaa agaataaaaa
     4681 aagataaaaa gaatagatcc cagccctgtg tataactcac tactttagtc agttccgcag
     4741 tattacaaaa ggatgtcgca aacgctgttt gctcctctac aaaacagacc ttaaaaccct
     4801 aaaggcttaa gtagcaccct cgcaagctcg gttgcggccg caatcgggca aatcgctgaa
     4861 tattcctttt gtctccgacc atcaggcacc tgagtcgctg tctttttcgt gacattcagt
     4921 tcgctgcgct cacggctctg gcagtgaatg ggggtaaatg gcactacagg cgccttttat
     4981 ggattcatgc aaggaaacta cccataatac aagaaaagcc cgtcacgggc ttctcagggc
     5041 gttttatggc gggtctgcta tgtggtgcta tctgactttt tgctgttcag cagttcctgc
     5101 cctctgattt tccagtctga ccacttcgga ttatcccgtg acaggtcatt cagactggct
     5161 aatgcaccca gtaaggcagc ggtatcatca acggggtctg acgctcagtg gaacgaaaac
     5221 tcacgttaag ggattttggt catgagatta tcagctttcg ctaaggatga tttctggaat
     5281 tcttccctat cagtgataga gattgacatc cctatcagtg atagagatac tgagcaccgc
     5341 taatgctgcc ctttcccgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     5401 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaagcttggg cccgaacaaa
     5461 aactcatctc agaagaggat ctgaatagcg ccgtcgacca tcatcatcat catcattgag
     5521 tttaaacggt ctccagcttg gctgttttgg cggatgagag aagattttca gcctgataca
     5581 gattaaatca gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc
     5641 ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag
     5701 tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc
     5761 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaactgg atccttactc
     5821 gagtctagac tgcaggcgga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5881 atcaatctaa agtatatatg agtaaacttg gtctgctgac aggacattat ttgccgacta
     5941 ccttggtgat ctcgcctttc acgtagtgga caaattcttc caactgatct gcgcgcgagg
     6001 ccaagcgatc ttcttcttgt ccaagataag cctgtctagc ttcaagtatg acgggctgat
     6061 actgggccgg caggcgctcc attgcccagt cggcagcgac atccttcggc gcgattttgc
     6121 cggttactgc gctgtaccaa atgcgggaca acgtaagcac tacatttcgc tcatcgccag
     6181 cccagtcggg cggcgagttc catagcgtta aggtttcatt tagcgcctca aatagatcct
     6241 gttcaggaac cggatcaaag agttcctccg ccgctggacc taccaaggca acgctatgtt
     6301 ctcttgcttt tgtcagcaag atagccagat caatgtcgat cgtggctggc tcgaagatac
     6361 ctgcaagaat gtcattgcgc tgccattctc caaattgcag ttcgcgctta gctggataac
     6421 gccacggaat gatgtcgtcg tgcacaacaa tggtgacttc tacagcgcgg agaatctcgc
     6481 tctctccagg ggaagccgaa gtttccaaaa ggtcgttgat caaagctcgc cgcgttgttt
     6541 catcaagcct tacggtcacc gtaaccagca aatcaatatc actgtgtggc ttcaggccgc
     6601 catccactgc ggagccgtac aaatgtacgg ccagcaacgt cggttcgaga tggcgctcga
     6661 tgacgccaac tacctctgat agttgagtcg atacttcggc gatcaccgct tccctcatac
     6721 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6781 tatttgaatg tatttagaaa aataaacaaa tagctagctc actcggtcgc taccagggtt
     6841 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6901 cgcgcacatt tccccgaaaa gtgccacc
//
