LOCUS       sequence-312955-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains arabinose-induced lambda Red and a tet-inducible gRNA 
            targeting fsaA..
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-fsaA
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    102..120
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    127..145
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     rep_origin      711..933
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             981..1931
                     /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(2054..2298)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(2299..2979)
                     /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(2976..3761)
                     /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(3767..4183)
                     /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             4192..4200
                     /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(4227..4511)
                     /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(4285..4304)
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             4538..5416
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        5547..5638
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5639..6430
                     /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(6569..6615)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     6768..6785
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(6841..6858)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(6874..6903)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_RNA        complement(join(6924..6928,1..71))
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 gactcggtgc cactttttca agttgataac ggactagcct tattttaact tgctatttct
       61 agctctaaaa cctgtggcga agtttgagca ggtgctcagt atctctatca ctgataggga
      121 tgtcaatctc tatcactgat agggaagaat tccagaaatc atccttagcg aaagctgata
      181 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgttg
      241 atgataccgc tgccttactg ggtgcattag ccagtctgaa tgacctgtca cgggataatc
      301 cgaagtggtc agactggaaa atcagagggc aggaactgct gaacagcaaa aagtcagata
      361 gcaccacata gcagacccgc cataaaacgc cctgagaagc ccgtgacggg cttttcttgt
      421 attatgggta gtttccttgc atgaatccat aaaaggcgcc tgtagtgcca tttaccccca
      481 ttcactgcca gagccgtgag cgcagcgaac tgaatgtcac gaaaaagaca gcgactcagg
      541 tgcctgatgg tcggagacaa aaggaatatt cagcgatttg cccgattgcg gccgcaaccg
      601 agcttgcgag ggtgctactt aagcctttag ggttttaagg tctgttttgt agaggagcaa
      661 acagcgtttg cgacatcctt ttgtaatact gcggaactga ctaaagtagt gagttataca
      721 cagggctggg atctattctt tttatctttt tttattcttt ctttattcta taaattataa
      781 ccacttgaat ataaacaaaa aaaacacaca aaggtctagc ggaatttaca gagggtctag
      841 cagaatttac aagttttcca gcaaaggtct agcagaattt acagataccc acaactcaaa
      901 ggaaaaggac tagtaattat cattgactag cccatctcaa ttggtatagt gattaaaatc
      961 acctagacca attgagatgt atgtctgaat tagttgtttt caaagcaaat gaactagcga
     1021 ttagtcgcta tgacttaacg gagcatgaaa ccaagctaat tttatgctgt gtggcactac
     1081 tcaaccccac gattgaaaac cctacaagga aagaacggac ggtatcgttc acttataacc
     1141 aatacgttca gatgatgaac atcagtaggg aaaatgctta tggtgtatta gctaaagcaa
     1201 ccagagagct gatgacgaga actgtggaaa tcaggaatcc tttggttaaa ggctttgaga
     1261 ttttccagtg gacaaactat gccaagttct caagcgaaaa attagaatta gtttttagtg
     1321 aagagatatt gccttatctt ttccagttaa aaaaattcat aaaatataat ctggaacatg
     1381 ttaagtcttt tgaaaacaaa tactctatga ggatttatga gtggttatta aaagaactaa
     1441 cacaaaagaa aactcacaag gcaaatatag agattagcct tgatgaattt aagttcatgt
     1501 taatgcttga aaataactac catgagttta aaaggcttaa ccaatgggtt ttgaaaccaa
     1561 taagtaaaga tttaaacact tacagcaata tgaaattggt ggttgataag cgaggccgcc
     1621 cgactgatac gttgattttc caagttgaac tagatagaca aatggatctc gtaaccgaac
     1681 ttgagaacaa ccagataaaa atgaatggtg acaaaatacc aacaaccatt acatcagatt
     1741 cctacctaca taacggacta agaaaaacac tacacgatgc tttaactgca aaaattcagc
     1801 tcaccagttt tgaggcaaaa tttttgagtg acatgcaaag taagtatgat ctcaatggtt
     1861 cgttctcatg gctcacgcaa aaacaacgaa ccacactaga gaacatactg gctaaatacg
     1921 gaaggatctg aggttcttat ggctcttgta tctatcagtg aagcatcaag actaacaaac
     1981 aaaagtagaa caactgttca ccgttacata tcaaagggaa aactgtccat acccatggat
     2041 tcttcgtctg tttctactgg tattggcaca aacctgattc caatttgagc aaggctatgt
     2101 gccatctcga tactcgttct taactcaaca gaagatgctt tgtgcataca gcccctcgtt
     2161 tattatttat ctcctcagcc agccgctgtg ctttcagtgg atttcggata acagaaaggc
     2221 cgggaaatac ccagcctcgc tttgtaacgg agtagacgaa agtgattgcg cctacccgga
     2281 tattatcgtg aggatgcgtc atcgccattg ctccccaaat acaaaaccaa tttcagccag
     2341 tgcctcgtcc attttttcga tgaactccgg cacgatctcg tcaaaactcg ccatgtactt
     2401 ttcatcccgc tcaatcacga cataatgcag gccttcacgc ttcatacgcg ggtcatagtt
     2461 ggcaaagtac caggcatttt ttcgcgtcac ccacatgctg tactgcacct gggccatgta
     2521 agctgacttt atggcctcga aaccaccgag ccggaacttc atgaaatccc gggaggtaaa
     2581 cgggcatttc agttcaaggc cgttgccgtc actgcataaa ccatcgggag agcaggcggt
     2641 acgcatactt tcgtcgcgat agatgatcgg ggattcagta acattcacgc cggaagtgaa
     2701 ttcaaacagg gttctggcgt cgttctcgta ctgttttccc caggccagtg ctttagcgtt
     2761 aacttccgga gccacaccgg tgcaaacctc agcaagcagg gtgtggaagt aggacatttt
     2821 catgtcaggc cacttctttc cggagcgggg ttttgctatc acgttgtgaa cttctgaagc
     2881 ggtgatgacg ccgagccgta atttgtgcca cgcatcatcc ccctgttcga cagctctcac
     2941 atcgatcccg gtacgctgca ggataatgtc cggtgtcatg ctgccacctt ctgctctgcg
     3001 gctttctgtt tcaggaatcc aagagctttt actgcttcgg cctgtgtcag ttctgacgat
     3061 gcacgaatgt cgcggcgaaa tatctgggaa cagagcggca ataagtcgtc atcccatgtt
     3121 ttatccaggg cgatcagcag agtgttaatc tcctgcatgg tttcatcgtt aaccggagtg
     3181 atgtcgcgtt ccggctgacg ttctgcagtg tatgcagtat tttcgacaat gcgctcggct
     3241 tcatccttgt catagatacc agcaaatccg aaggccagac gggcacactg aatcatggct
     3301 ttatgacgta acatccgttt gggatgcgac tgccacggcc ccgtgatttc tctgccttcg
     3361 cgagttttga atggttcgcg gcggcattca tccatccatt cggtaacgca gatcggatga
     3421 ttacggtcct tgcggtaaat ccggcatgta caggattcat tgtcctgctc aaagtccatg
     3481 ccatcaaact gctggttttc attgatgatg cgggaccagc catcaacgcc caccaccgga
     3541 acgatgccat tctgcttatc aggaaaggcg taaatttctt tcgtccacgg attaaggccg
     3601 tactggttgg caacgatcag taatgcgatg aactgcgcat cgctggcatc acctttaaat
     3661 gccgtctggc gaagagtggt gatcagttcc tgtgggtcga cagaatccat gccgacacgt
     3721 tcagccagct tcccagccag cgttgcgagt gcagtactca ttcgttttat acctctgaat
     3781 caatatcaac ctggtggtga gcaatggttt caaccatgta ccggatgtgt tctgccatgc
     3841 gctcctgaaa ctcaacatcg tcatcaaacg cacgggtaat ggattttttg ctggccccgt
     3901 ggcgttgcaa atgatcgatg catagcgatt caaacaggtg ctggggcagg cctttttcca
     3961 tgtcgtctgc cagttctgcc tctttctctt cacgggcgag ctgctggtag tgacgcgccc
     4021 agctctgagc ctcaagacga tcctgaatgt aataagcgtt catggctgaa ctcctgaaat
     4081 agctgtgaaa atatcgcccg cgaaatgccg ggctgattag gaaaacagga aagggggtta
     4141 gtgaatgctt ttgcttgatc tcagtttcag tattaatatc cattttttat aacctcctta
     4201 gagctcgaat tcccaaaaaa acgggtatgg agaaacagta gagagttgcg ataaaaagcg
     4261 tcaggtagga tccgctaatc ttatggataa aaatgctatg gcatagcaaa gtgtgacgcc
     4321 gtgcaaataa tcaatgtgga cttttctgcc gtgattatag acacttttgt tacgcgtttt
     4381 tgtcatggct ttggtcccgc tttgttacag aatgctttta ataagcgggg ttaccggttt
     4441 ggttagcgag aagagccagt aaaagacgca gtgacggcaa tgtctgatgc aatatggaca
     4501 attggtttct tctctgaatg gcgggagtat gaaaagtatg gctgaagcgc aaaatgatcc
     4561 cctgctgccg ggatactcgt ttaatgccca tctggtggcg ggtttaacgc cgattgaggc
     4621 caacggttat ctcgattttt ttatcgaccg accgctggga atgaaaggtt atattctcaa
     4681 tctcaccatt cgcggtcagg gggtggtgaa aaatcaggga cgagaatttg tttgccgacc
     4741 gggtgatatt ttgctgttcc cgccaggaga gattcatcac tacggtcgtc atccggaggc
     4801 tcgcgaatgg tatcaccagt gggtttactt tcgtccgcgc gcctactggc atgaatggct
     4861 taactggccg tcaatatttg ccaatacggg gttctttcgc ccggatgaag cgcaccagcc
     4921 gcatttcagc gacctgtttg ggcaaatcat taacgccggg caaggggaag ggcgctattc
     4981 ggagctgctg gcgataaatc tgcttgagca attgttactg cggcgcatgg aagcgattaa
     5041 cgagtcgctc catccaccga tggataatcg ggtacgcgag gcttgtcagt acatcagcga
     5101 tcacctggca gacagcaatt ttgatatcgc cagcgtcgca cagcatgttt gcttgtcgcc
     5161 gtcgcgtctg tcacatcttt tccgccagca gttagggatt agcgtcttaa gctggcgcga
     5221 ggaccaacgt atcagccagg cgaagctgct tttgagcacc acccggatgc ctatcgccac
     5281 cgtcggtcgc aatgttggtt ttgacgatca actctacttc tcgcgggtat ttaaaaaatg
     5341 caccggggcc agcccgagcg agttccgtgc cggttgtgaa gaaaaagtga atgatgtagc
     5401 cgtcaagttg tcataataaa tcgatgcagg tggcactttt cggggaaatg tgcgcggaac
     5461 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     5521 ctggtagcga ccgagtgagc tagctatttg tttatttttc taaatacatt caaatatgta
     5581 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5641 gagggaagcg gtgatcgccg aagtatcgac tcaactatca gaggtagttg gcgtcatcga
     5701 gcgccatctc gaaccgacgt tgctggccgt acatttgtac ggctccgcag tggatggcgg
     5761 cctgaagcca cacagtgata ttgatttgct ggttacggtg accgtaaggc ttgatgaaac
     5821 aacgcggcga gctttgatca acgacctttt ggaaacttcg gcttcccctg gagagagcga
     5881 gattctccgc gctgtagaag tcaccattgt tgtgcacgac gacatcattc cgtggcgtta
     5941 tccagctaag cgcgaactgc aatttggaga atggcagcgc aatgacattc ttgcaggtat
     6001 cttcgagcca gccacgatcg acattgatct ggctatcttg ctgacaaaag caagagaaca
     6061 tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt gatccggttc ctgaacagga
     6121 tctatttgag gcgctaaatg aaaccttaac gctatggaac tcgccgcccg actgggctgg
     6181 cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa
     6241 aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg cccagtatca
     6301 gcccgtcata cttgaagcta gacaggctta tcttggacaa gaagaagatc gcttggcctc
     6361 gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa ggcgagatca ccaaggtagt
     6421 cggcaaataa tgtcctgtca gcagaccaag tttactcata tatactttag attgatttaa
     6481 aacttcattt ttaatttaaa aggatctagg tgaagatccg cctgcagtct agactcgagt
     6541 aaggatccag ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc
     6601 ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac cccacactac
     6661 catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg accaccgcgc
     6721 tactgccgcc aggcaaattc tgttttatca gaccgcttct gcgttctgat ttaatctgta
     6781 tcaggctgaa aatcttctct catccgccaa aacagccaag ctggagaccg tttaaactca
     6841 atgatgatga tgatgatggt cgacggcgct attcagatcc tcttctgaga tgagtttttg
     6901 ttcgggccca agcttcaaaa aaagcacc
//
