LOCUS       sequence-182545-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains arabinose-induced lambda Red and a tet-inducible gRNA 
            targeting hisG..
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-hisG
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      27..249
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     protein_bind    815..833
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    840..858
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        889..964
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             985..1014
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1030..1047
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1103..1120)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1273..1319
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1458..2249)
                     /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(2250..2341)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(2472..3350)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        3377..3661
                     /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     3584..3603
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             3688..3696
                     /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             3705..4121
                     /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             4127..4912
                     /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             4909..5589
                     /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      5590..5834
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(5957..6907)
                     /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"
ORIGIN
        1 attttaatca ctataccaat tgagatgggc tagtcaatga taattactag tccttttcct
       61 ttgagttgtg ggtatctgta aattctgcta gacctttgct ggaaaacttg taaattctgc
      121 tagaccctct gtaaattccg ctagaccttt gtgtgttttt tttgtttata ttcaagtggt
      181 tataatttat agaataaaga aagaataaaa aaagataaaa agaatagatc ccagccctgt
      241 gtataactca ctactttagt cagttccgca gtattacaaa aggatgtcgc aaacgctgtt
      301 tgctcctcta caaaacagac cttaaaaccc taaaggctta agtagcaccc tcgcaagctc
      361 ggttgcggcc gcaatcgggc aaatcgctga atattccttt tgtctccgac catcaggcac
      421 ctgagtcgct gtctttttcg tgacattcag ttcgctgcgc tcacggctct ggcagtgaat
      481 gggggtaaat ggcactacag gcgcctttta tggattcatg caaggaaact acccataata
      541 caagaaaagc ccgtcacggg cttctcaggg cgttttatgg cgggtctgct atgtggtgct
      601 atctgacttt ttgctgttca gcagttcctg ccctctgatt ttccagtctg accacttcgg
      661 attatcccgt gacaggtcat tcagactggc taatgcaccc agtaaggcag cggtatcatc
      721 aacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
      781 atcagctttc gctaaggatg atttctggaa ttcttcccta tcagtgatag agattgacat
      841 ccctatcagt gatagagata ctgagcacgc cgaaatccag acgacgcagt tttagagcta
      901 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
      961 gtgctttttt tgaagcttgg gcccgaacaa aaactcatct cagaagagga tctgaatagc
     1021 gccgtcgacc atcatcatca tcatcattga gtttaaacgg tctccagctt ggctgttttg
     1081 gcggatgaga gaagattttc agcctgatac agattaaatc agaacgcaga agcggtctga
     1141 taaaacagaa tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact
     1201 cagaagtgaa acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga
     1261 actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
     1321 tgttgtttgt cggtgaactg gatccttact cgagtctaga ctgcaggcgg atcttcacct
     1381 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     1441 ggtctgctga caggacatta tttgccgact accttggtga tctcgccttt cacgtagtgg
     1501 acaaattctt ccaactgatc tgcgcgcgag gccaagcgat cttcttcttg tccaagataa
     1561 gcctgtctag cttcaagtat gacgggctga tactgggccg gcaggcgctc cattgcccag
     1621 tcggcagcga catccttcgg cgcgattttg ccggttactg cgctgtacca aatgcgggac
     1681 aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt
     1741 aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc
     1801 gccgctggac ctaccaaggc aacgctatgt tctcttgctt ttgtcagcaa gatagccaga
     1861 tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa tgtcattgcg ctgccattct
     1921 ccaaattgca gttcgcgctt agctggataa cgccacggaa tgatgtcgtc gtgcacaaca
     1981 atggtgactt ctacagcgcg gagaatctcg ctctctccag gggaagccga agtttccaaa
     2041 aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc
     2101 aaatcaatat cactgtgtgg cttcaggccg ccatccactg cggagccgta caaatgtacg
     2161 gccagcaacg tcggttcgag atggcgctcg atgacgccaa ctacctctga tagttgagtc
     2221 gatacttcgg cgatcaccgc ttccctcata ctcttccttt ttcaatatta ttgaagcatt
     2281 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     2341 atagctagct cactcggtcg ctaccagggt tattgtctca tgagcggata catatttgaa
     2401 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     2461 gcatcgattt attatgacaa cttgacggct acatcattca ctttttcttc acaaccggca
     2521 cggaactcgc tcgggctggc cccggtgcat tttttaaata cccgcgagaa gtagagttga
     2581 tcgtcaaaac caacattgcg accgacggtg gcgataggca tccgggtggt gctcaaaagc
     2641 agcttcgcct ggctgatacg ttggtcctcg cgccagctta agacgctaat ccctaactgc
     2701 tggcggaaaa gatgtgacag acgcgacggc gacaagcaaa catgctgtgc gacgctggcg
     2761 atatcaaaat tgctgtctgc caggtgatcg ctgatgtact gacaagcctc gcgtacccga
     2821 ttatccatcg gtggatggag cgactcgtta atcgcttcca tgcgccgcag taacaattgc
     2881 tcaagcagat ttatcgccag cagctccgaa tagcgccctt ccccttgccc ggcgttaatg
     2941 atttgcccaa acaggtcgct gaaatgcggc tggtgcgctt catccgggcg aaagaacccc
     3001 gtattggcaa atattgacgg ccagttaagc cattcatgcc agtaggcgcg cggacgaaag
     3061 taaacccact ggtgatacca ttcgcgagcc tccggatgac gaccgtagtg atgaatctct
     3121 cctggcggga acagcaaaat atcacccggt cggcaaacaa attctcgtcc ctgatttttc
     3181 accaccccct gaccgcgaat ggtgagattg agaatataac ctttcattcc cagcggtcgg
     3241 tcgataaaaa aatcgagata accgttggcc tcaatcggcg ttaaacccgc caccagatgg
     3301 gcattaaacg agtatcccgg cagcagggga tcattttgcg cttcagccat acttttcata
     3361 ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg ccgtcactgc
     3421 gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa aagcattctg
     3481 taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta taatcacggc
     3541 agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat agcattttta
     3601 tccataagat tagcggatcc tacctgacgc tttttatcgc aactctctac tgtttctcca
     3661 tacccgtttt tttgggaatt cgagctctaa ggaggttata aaaaatggat attaatactg
     3721 aaactgagat caagcaaaag cattcactaa ccccctttcc tgttttccta atcagcccgg
     3781 catttcgcgg gcgatatttt cacagctatt tcaggagttc agccatgaac gcttattaca
     3841 ttcaggatcg tcttgaggct cagagctggg cgcgtcacta ccagcagctc gcccgtgaag
     3901 agaaagaggc agaactggca gacgacatgg aaaaaggcct gccccagcac ctgtttgaat
     3961 cgctatgcat cgatcatttg caacgccacg gggccagcaa aaaatccatt acccgtgcgt
     4021 ttgatgacga tgttgagttt caggagcgca tggcagaaca catccggtac atggttgaaa
     4081 ccattgctca ccaccaggtt gatattgatt cagaggtata aaacgaatga gtactgcact
     4141 cgcaacgctg gctgggaagc tggctgaacg tgtcggcatg gattctgtcg acccacagga
     4201 actgatcacc actcttcgcc agacggcatt taaaggtgat gccagcgatg cgcagttcat
     4261 cgcattactg atcgttgcca accagtacgg ccttaatccg tggacgaaag aaatttacgc
     4321 ctttcctgat aagcagaatg gcatcgttcc ggtggtgggc gttgatggct ggtcccgcat
     4381 catcaatgaa aaccagcagt ttgatggcat ggactttgag caggacaatg aatcctgtac
     4441 atgccggatt taccgcaagg accgtaatca tccgatctgc gttaccgaat ggatggatga
     4501 atgccgccgc gaaccattca aaactcgcga aggcagagaa atcacggggc cgtggcagtc
     4561 gcatcccaaa cggatgttac gtcataaagc catgattcag tgtgcccgtc tggccttcgg
     4621 atttgctggt atctatgaca aggatgaagc cgagcgcatt gtcgaaaata ctgcatacac
     4681 tgcagaacgt cagccggaac gcgacatcac tccggttaac gatgaaacca tgcaggagat
     4741 taacactctg ctgatcgccc tggataaaac atgggatgac gacttattgc cgctctgttc
     4801 ccagatattt cgccgcgaca ttcgtgcatc gtcagaactg acacaggccg aagcagtaaa
     4861 agctcttgga ttcctgaaac agaaagccgc agagcagaag gtggcagcat gacaccggac
     4921 attatcctgc agcgtaccgg gatcgatgtg agagctgtcg aacaggggga tgatgcgtgg
     4981 cacaaattac ggctcggcgt catcaccgct tcagaagttc acaacgtgat agcaaaaccc
     5041 cgctccggaa agaagtggcc tgacatgaaa atgtcctact tccacaccct gcttgctgag
     5101 gtttgcaccg gtgtggctcc ggaagttaac gctaaagcac tggcctgggg aaaacagtac
     5161 gagaacgacg ccagaaccct gtttgaattc acttccggcg tgaatgttac tgaatccccg
     5221 atcatctatc gcgacgaaag tatgcgtacc gcctgctctc ccgatggttt atgcagtgac
     5281 ggcaacggcc ttgaactgaa atgcccgttt acctcccggg atttcatgaa gttccggctc
     5341 ggtggtttcg aggccataaa gtcagcttac atggcccagg tgcagtacag catgtgggtg
     5401 acgcgaaaaa atgcctggta ctttgccaac tatgacccgc gtatgaagcg tgaaggcctg
     5461 cattatgtcg tgattgagcg ggatgaaaag tacatggcga gttttgacga gatcgtgccg
     5521 gagttcatcg aaaaaatgga cgaggcactg gctgaaattg gttttgtatt tggggagcaa
     5581 tggcgatgac gcatcctcac gataatatcc gggtaggcgc aatcactttc gtctactccg
     5641 ttacaaagcg aggctgggta tttcccggcc tttctgttat ccgaaatcca ctgaaagcac
     5701 agcggctggc tgaggagata aataataaac gaggggctgt atgcacaaag catcttctgt
     5761 tgagttaaga acgagtatcg agatggcaca tagccttgct caaattggaa tcaggtttgt
     5821 gccaatacca gtagaaacag acgaagaatc catgggtatg gacagttttc cctttgatat
     5881 gtaacggtga acagttgttc tacttttgtt tgttagtctt gatgcttcac tgatagatac
     5941 aagagccata agaacctcag atccttccgt atttagccag tatgttctct agtgtggttc
     6001 gttgtttttg cgtgagccat gagaacgaac cattgagatc atacttactt tgcatgtcac
     6061 tcaaaaattt tgcctcaaaa ctggtgagct gaatttttgc agttaaagca tcgtgtagtg
     6121 tttttcttag tccgttatgt aggtaggaat ctgatgtaat ggttgttggt attttgtcac
     6181 cattcatttt tatctggttg ttctcaagtt cggttacgag atccatttgt ctatctagtt
     6241 caacttggaa aatcaacgta tcagtcgggc ggcctcgctt atcaaccacc aatttcatat
     6301 tgctgtaagt gtttaaatct ttacttattg gtttcaaaac ccattggtta agccttttaa
     6361 actcatggta gttattttca agcattaaca tgaacttaaa ttcatcaagg ctaatctcta
     6421 tatttgcctt gtgagttttc ttttgtgtta gttcttttaa taaccactca taaatcctca
     6481 tagagtattt gttttcaaaa gacttaacat gttccagatt atattttatg aattttttta
     6541 actggaaaag ataaggcaat atctcttcac taaaaactaa ttctaatttt tcgcttgaga
     6601 acttggcata gtttgtccac tggaaaatct caaagccttt aaccaaagga ttcctgattt
     6661 ccacagttct cgtcatcagc tctctggttg ctttagctaa tacaccataa gcattttccc
     6721 tactgatgtt catcatctga acgtattggt tataagtgaa cgataccgtc cgttctttcc
     6781 ttgtagggtt ttcaatcgtg gggttgagta gtgccacaca gcataaaatt agcttggttt
     6841 catgctccgt taagtcatag cgactaatcg ctagttcatt tgctttgaaa acaactaatt
     6901 cagacataca tctcaattgg tctaggtg
//
