LOCUS       sequence-201662-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains arabinose-induced lambda Red and a tet-inducible gRNA 
            targeting ompT..
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-ompT
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    72..90
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    97..115
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     rep_origin      681..903
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             951..1901
                     /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"
     terminator      complement(2024..2268)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(2269..2949)
                     /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"
     CDS             complement(2946..3731)
                     /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             complement(3737..4153)
                     /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"
     RBS             4162..4170
                     /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)"
     promoter        complement(4197..4481)
                     /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     complement(4255..4274)
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             4508..5386
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        5517..5608
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5609..6400
                     /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"
     terminator      complement(6539..6585)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     6738..6755
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(6811..6828)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(6844..6873)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_RNA        complement(join(6894..6928,1..41))
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 ggactagcct tattttaact tgctatttct agctctaaaa caaaagtcag tcaactcgac
       61 tgtgctcagt atctctatca ctgataggga tgtcaatctc tatcactgat agggaagaat
      121 tccagaaatc atccttagcg aaagctgata atctcatgac caaaatccct taacgtgagt
      181 tttcgttcca ctgagcgtca gaccccgttg atgataccgc tgccttactg ggtgcattag
      241 ccagtctgaa tgacctgtca cgggataatc cgaagtggtc agactggaaa atcagagggc
      301 aggaactgct gaacagcaaa aagtcagata gcaccacata gcagacccgc cataaaacgc
      361 cctgagaagc ccgtgacggg cttttcttgt attatgggta gtttccttgc atgaatccat
      421 aaaaggcgcc tgtagtgcca tttaccccca ttcactgcca gagccgtgag cgcagcgaac
      481 tgaatgtcac gaaaaagaca gcgactcagg tgcctgatgg tcggagacaa aaggaatatt
      541 cagcgatttg cccgattgcg gccgcaaccg agcttgcgag ggtgctactt aagcctttag
      601 ggttttaagg tctgttttgt agaggagcaa acagcgtttg cgacatcctt ttgtaatact
      661 gcggaactga ctaaagtagt gagttataca cagggctggg atctattctt tttatctttt
      721 tttattcttt ctttattcta taaattataa ccacttgaat ataaacaaaa aaaacacaca
      781 aaggtctagc ggaatttaca gagggtctag cagaatttac aagttttcca gcaaaggtct
      841 agcagaattt acagataccc acaactcaaa ggaaaaggac tagtaattat cattgactag
      901 cccatctcaa ttggtatagt gattaaaatc acctagacca attgagatgt atgtctgaat
      961 tagttgtttt caaagcaaat gaactagcga ttagtcgcta tgacttaacg gagcatgaaa
     1021 ccaagctaat tttatgctgt gtggcactac tcaaccccac gattgaaaac cctacaagga
     1081 aagaacggac ggtatcgttc acttataacc aatacgttca gatgatgaac atcagtaggg
     1141 aaaatgctta tggtgtatta gctaaagcaa ccagagagct gatgacgaga actgtggaaa
     1201 tcaggaatcc tttggttaaa ggctttgaga ttttccagtg gacaaactat gccaagttct
     1261 caagcgaaaa attagaatta gtttttagtg aagagatatt gccttatctt ttccagttaa
     1321 aaaaattcat aaaatataat ctggaacatg ttaagtcttt tgaaaacaaa tactctatga
     1381 ggatttatga gtggttatta aaagaactaa cacaaaagaa aactcacaag gcaaatatag
     1441 agattagcct tgatgaattt aagttcatgt taatgcttga aaataactac catgagttta
     1501 aaaggcttaa ccaatgggtt ttgaaaccaa taagtaaaga tttaaacact tacagcaata
     1561 tgaaattggt ggttgataag cgaggccgcc cgactgatac gttgattttc caagttgaac
     1621 tagatagaca aatggatctc gtaaccgaac ttgagaacaa ccagataaaa atgaatggtg
     1681 acaaaatacc aacaaccatt acatcagatt cctacctaca taacggacta agaaaaacac
     1741 tacacgatgc tttaactgca aaaattcagc tcaccagttt tgaggcaaaa tttttgagtg
     1801 acatgcaaag taagtatgat ctcaatggtt cgttctcatg gctcacgcaa aaacaacgaa
     1861 ccacactaga gaacatactg gctaaatacg gaaggatctg aggttcttat ggctcttgta
     1921 tctatcagtg aagcatcaag actaacaaac aaaagtagaa caactgttca ccgttacata
     1981 tcaaagggaa aactgtccat acccatggat tcttcgtctg tttctactgg tattggcaca
     2041 aacctgattc caatttgagc aaggctatgt gccatctcga tactcgttct taactcaaca
     2101 gaagatgctt tgtgcataca gcccctcgtt tattatttat ctcctcagcc agccgctgtg
     2161 ctttcagtgg atttcggata acagaaaggc cgggaaatac ccagcctcgc tttgtaacgg
     2221 agtagacgaa agtgattgcg cctacccgga tattatcgtg aggatgcgtc atcgccattg
     2281 ctccccaaat acaaaaccaa tttcagccag tgcctcgtcc attttttcga tgaactccgg
     2341 cacgatctcg tcaaaactcg ccatgtactt ttcatcccgc tcaatcacga cataatgcag
     2401 gccttcacgc ttcatacgcg ggtcatagtt ggcaaagtac caggcatttt ttcgcgtcac
     2461 ccacatgctg tactgcacct gggccatgta agctgacttt atggcctcga aaccaccgag
     2521 ccggaacttc atgaaatccc gggaggtaaa cgggcatttc agttcaaggc cgttgccgtc
     2581 actgcataaa ccatcgggag agcaggcggt acgcatactt tcgtcgcgat agatgatcgg
     2641 ggattcagta acattcacgc cggaagtgaa ttcaaacagg gttctggcgt cgttctcgta
     2701 ctgttttccc caggccagtg ctttagcgtt aacttccgga gccacaccgg tgcaaacctc
     2761 agcaagcagg gtgtggaagt aggacatttt catgtcaggc cacttctttc cggagcgggg
     2821 ttttgctatc acgttgtgaa cttctgaagc ggtgatgacg ccgagccgta atttgtgcca
     2881 cgcatcatcc ccctgttcga cagctctcac atcgatcccg gtacgctgca ggataatgtc
     2941 cggtgtcatg ctgccacctt ctgctctgcg gctttctgtt tcaggaatcc aagagctttt
     3001 actgcttcgg cctgtgtcag ttctgacgat gcacgaatgt cgcggcgaaa tatctgggaa
     3061 cagagcggca ataagtcgtc atcccatgtt ttatccaggg cgatcagcag agtgttaatc
     3121 tcctgcatgg tttcatcgtt aaccggagtg atgtcgcgtt ccggctgacg ttctgcagtg
     3181 tatgcagtat tttcgacaat gcgctcggct tcatccttgt catagatacc agcaaatccg
     3241 aaggccagac gggcacactg aatcatggct ttatgacgta acatccgttt gggatgcgac
     3301 tgccacggcc ccgtgatttc tctgccttcg cgagttttga atggttcgcg gcggcattca
     3361 tccatccatt cggtaacgca gatcggatga ttacggtcct tgcggtaaat ccggcatgta
     3421 caggattcat tgtcctgctc aaagtccatg ccatcaaact gctggttttc attgatgatg
     3481 cgggaccagc catcaacgcc caccaccgga acgatgccat tctgcttatc aggaaaggcg
     3541 taaatttctt tcgtccacgg attaaggccg tactggttgg caacgatcag taatgcgatg
     3601 aactgcgcat cgctggcatc acctttaaat gccgtctggc gaagagtggt gatcagttcc
     3661 tgtgggtcga cagaatccat gccgacacgt tcagccagct tcccagccag cgttgcgagt
     3721 gcagtactca ttcgttttat acctctgaat caatatcaac ctggtggtga gcaatggttt
     3781 caaccatgta ccggatgtgt tctgccatgc gctcctgaaa ctcaacatcg tcatcaaacg
     3841 cacgggtaat ggattttttg ctggccccgt ggcgttgcaa atgatcgatg catagcgatt
     3901 caaacaggtg ctggggcagg cctttttcca tgtcgtctgc cagttctgcc tctttctctt
     3961 cacgggcgag ctgctggtag tgacgcgccc agctctgagc ctcaagacga tcctgaatgt
     4021 aataagcgtt catggctgaa ctcctgaaat agctgtgaaa atatcgcccg cgaaatgccg
     4081 ggctgattag gaaaacagga aagggggtta gtgaatgctt ttgcttgatc tcagtttcag
     4141 tattaatatc cattttttat aacctcctta gagctcgaat tcccaaaaaa acgggtatgg
     4201 agaaacagta gagagttgcg ataaaaagcg tcaggtagga tccgctaatc ttatggataa
     4261 aaatgctatg gcatagcaaa gtgtgacgcc gtgcaaataa tcaatgtgga cttttctgcc
     4321 gtgattatag acacttttgt tacgcgtttt tgtcatggct ttggtcccgc tttgttacag
     4381 aatgctttta ataagcgggg ttaccggttt ggttagcgag aagagccagt aaaagacgca
     4441 gtgacggcaa tgtctgatgc aatatggaca attggtttct tctctgaatg gcgggagtat
     4501 gaaaagtatg gctgaagcgc aaaatgatcc cctgctgccg ggatactcgt ttaatgccca
     4561 tctggtggcg ggtttaacgc cgattgaggc caacggttat ctcgattttt ttatcgaccg
     4621 accgctggga atgaaaggtt atattctcaa tctcaccatt cgcggtcagg gggtggtgaa
     4681 aaatcaggga cgagaatttg tttgccgacc gggtgatatt ttgctgttcc cgccaggaga
     4741 gattcatcac tacggtcgtc atccggaggc tcgcgaatgg tatcaccagt gggtttactt
     4801 tcgtccgcgc gcctactggc atgaatggct taactggccg tcaatatttg ccaatacggg
     4861 gttctttcgc ccggatgaag cgcaccagcc gcatttcagc gacctgtttg ggcaaatcat
     4921 taacgccggg caaggggaag ggcgctattc ggagctgctg gcgataaatc tgcttgagca
     4981 attgttactg cggcgcatgg aagcgattaa cgagtcgctc catccaccga tggataatcg
     5041 ggtacgcgag gcttgtcagt acatcagcga tcacctggca gacagcaatt ttgatatcgc
     5101 cagcgtcgca cagcatgttt gcttgtcgcc gtcgcgtctg tcacatcttt tccgccagca
     5161 gttagggatt agcgtcttaa gctggcgcga ggaccaacgt atcagccagg cgaagctgct
     5221 tttgagcacc acccggatgc ctatcgccac cgtcggtcgc aatgttggtt ttgacgatca
     5281 actctacttc tcgcgggtat ttaaaaaatg caccggggcc agcccgagcg agttccgtgc
     5341 cggttgtgaa gaaaaagtga atgatgtagc cgtcaagttg tcataataaa tcgatgcagg
     5401 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5461 aaatatgtat ccgctcatga gacaataacc ctggtagcga ccgagtgagc tagctatttg
     5521 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5581 gcttcaataa tattgaaaaa ggaagagtat gagggaagcg gtgatcgccg aagtatcgac
     5641 tcaactatca gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt
     5701 acatttgtac ggctccgcag tggatggcgg cctgaagcca cacagtgata ttgatttgct
     5761 ggttacggtg accgtaaggc ttgatgaaac aacgcggcga gctttgatca acgacctttt
     5821 ggaaacttcg gcttcccctg gagagagcga gattctccgc gctgtagaag tcaccattgt
     5881 tgtgcacgac gacatcattc cgtggcgtta tccagctaag cgcgaactgc aatttggaga
     5941 atggcagcgc aatgacattc ttgcaggtat cttcgagcca gccacgatcg acattgatct
     6001 ggctatcttg ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc cagcggcgga
     6061 ggaactcttt gatccggttc ctgaacagga tctatttgag gcgctaaatg aaaccttaac
     6121 gctatggaac tcgccgcccg actgggctgg cgatgagcga aatgtagtgc ttacgttgtc
     6181 ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg
     6241 ggcaatggag cgcctgccgg cccagtatca gcccgtcata cttgaagcta gacaggctta
     6301 tcttggacaa gaagaagatc gcttggcctc gcgcgcagat cagttggaag aatttgtcca
     6361 ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa tgtcctgtca gcagaccaag
     6421 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6481 tgaagatccg cctgcagtct agactcgagt aaggatccag ttcaccgaca aacaacagat
     6541 aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt atttgatgcc tggcagttcc
     6601 ctactctcgc atggggagac cccacactac catcggcgct acggcgtttc acttctgagt
     6661 tcggcatggg gtcaggtggg accaccgcgc tactgccgcc aggcaaattc tgttttatca
     6721 gaccgcttct gcgttctgat ttaatctgta tcaggctgaa aatcttctct catccgccaa
     6781 aacagccaag ctggagaccg tttaaactca atgatgatga tgatgatggt cgacggcgct
     6841 attcagatcc tcttctgaga tgagtttttg ttcgggccca agcttcaaaa aaagcaccga
     6901 ctcggtgcca ctttttcaag ttgataac
//
