LOCUS       sequence-319305-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains arabinose-induced lambda Red and a tet-inducible gRNA 
            targeting ldhA..
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-ldhA
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      334..556
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             604..1554
                     /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(1677..1921)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(1922..2602)
                     /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(2599..3384)
                     /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(3390..3806)
                     /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             3815..3823
                     /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(3850..4134)
                     /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(3908..3927)
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             4161..5039
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        5170..5261
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5262..6053
                     /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(6192..6238)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     6391..6408
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(6464..6481)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(6497..6526)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_RNA        complement(6547..6622)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    6653..6671
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6678..6696
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 cgccataaaa cgccctgaga agcccgtgac gggcttttct tgtattatgg gtagtttcct
       61 tgcatgaatc cataaaaggc gcctgtagtg ccatttaccc ccattcactg ccagagccgt
      121 gagcgcagcg aactgaatgt cacgaaaaag acagcgactc aggtgcctga tggtcggaga
      181 caaaaggaat attcagcgat ttgcccgatt gcggccgcaa ccgagcttgc gagggtgcta
      241 cttaagcctt tagggtttta aggtctgttt tgtagaggag caaacagcgt ttgcgacatc
      301 cttttgtaat actgcggaac tgactaaagt agtgagttat acacagggct gggatctatt
      361 ctttttatct ttttttattc tttctttatt ctataaatta taaccacttg aatataaaca
      421 aaaaaaacac acaaaggtct agcggaattt acagagggtc tagcagaatt tacaagtttt
      481 ccagcaaagg tctagcagaa tttacagata cccacaactc aaaggaaaag gactagtaat
      541 tatcattgac tagcccatct caattggtat agtgattaaa atcacctaga ccaattgaga
      601 tgtatgtctg aattagttgt tttcaaagca aatgaactag cgattagtcg ctatgactta
      661 acggagcatg aaaccaagct aattttatgc tgtgtggcac tactcaaccc cacgattgaa
      721 aaccctacaa ggaaagaacg gacggtatcg ttcacttata accaatacgt tcagatgatg
      781 aacatcagta gggaaaatgc ttatggtgta ttagctaaag caaccagaga gctgatgacg
      841 agaactgtgg aaatcaggaa tcctttggtt aaaggctttg agattttcca gtggacaaac
      901 tatgccaagt tctcaagcga aaaattagaa ttagttttta gtgaagagat attgccttat
      961 cttttccagt taaaaaaatt cataaaatat aatctggaac atgttaagtc ttttgaaaac
     1021 aaatactcta tgaggattta tgagtggtta ttaaaagaac taacacaaaa gaaaactcac
     1081 aaggcaaata tagagattag ccttgatgaa tttaagttca tgttaatgct tgaaaataac
     1141 taccatgagt ttaaaaggct taaccaatgg gttttgaaac caataagtaa agatttaaac
     1201 acttacagca atatgaaatt ggtggttgat aagcgaggcc gcccgactga tacgttgatt
     1261 ttccaagttg aactagatag acaaatggat ctcgtaaccg aacttgagaa caaccagata
     1321 aaaatgaatg gtgacaaaat accaacaacc attacatcag attcctacct acataacgga
     1381 ctaagaaaaa cactacacga tgctttaact gcaaaaattc agctcaccag ttttgaggca
     1441 aaatttttga gtgacatgca aagtaagtat gatctcaatg gttcgttctc atggctcacg
     1501 caaaaacaac gaaccacact agagaacata ctggctaaat acggaaggat ctgaggttct
     1561 tatggctctt gtatctatca gtgaagcatc aagactaaca aacaaaagta gaacaactgt
     1621 tcaccgttac atatcaaagg gaaaactgtc catacccatg gattcttcgt ctgtttctac
     1681 tggtattggc acaaacctga ttccaatttg agcaaggcta tgtgccatct cgatactcgt
     1741 tcttaactca acagaagatg ctttgtgcat acagcccctc gtttattatt tatctcctca
     1801 gccagccgct gtgctttcag tggatttcgg ataacagaaa ggccgggaaa tacccagcct
     1861 cgctttgtaa cggagtagac gaaagtgatt gcgcctaccc ggatattatc gtgaggatgc
     1921 gtcatcgcca ttgctcccca aatacaaaac caatttcagc cagtgcctcg tccatttttt
     1981 cgatgaactc cggcacgatc tcgtcaaaac tcgccatgta cttttcatcc cgctcaatca
     2041 cgacataatg caggccttca cgcttcatac gcgggtcata gttggcaaag taccaggcat
     2101 tttttcgcgt cacccacatg ctgtactgca cctgggccat gtaagctgac tttatggcct
     2161 cgaaaccacc gagccggaac ttcatgaaat cccgggaggt aaacgggcat ttcagttcaa
     2221 ggccgttgcc gtcactgcat aaaccatcgg gagagcaggc ggtacgcata ctttcgtcgc
     2281 gatagatgat cggggattca gtaacattca cgccggaagt gaattcaaac agggttctgg
     2341 cgtcgttctc gtactgtttt ccccaggcca gtgctttagc gttaacttcc ggagccacac
     2401 cggtgcaaac ctcagcaagc agggtgtgga agtaggacat tttcatgtca ggccacttct
     2461 ttccggagcg gggttttgct atcacgttgt gaacttctga agcggtgatg acgccgagcc
     2521 gtaatttgtg ccacgcatca tccccctgtt cgacagctct cacatcgatc ccggtacgct
     2581 gcaggataat gtccggtgtc atgctgccac cttctgctct gcggctttct gtttcaggaa
     2641 tccaagagct tttactgctt cggcctgtgt cagttctgac gatgcacgaa tgtcgcggcg
     2701 aaatatctgg gaacagagcg gcaataagtc gtcatcccat gttttatcca gggcgatcag
     2761 cagagtgtta atctcctgca tggtttcatc gttaaccgga gtgatgtcgc gttccggctg
     2821 acgttctgca gtgtatgcag tattttcgac aatgcgctcg gcttcatcct tgtcatagat
     2881 accagcaaat ccgaaggcca gacgggcaca ctgaatcatg gctttatgac gtaacatccg
     2941 tttgggatgc gactgccacg gccccgtgat ttctctgcct tcgcgagttt tgaatggttc
     3001 gcggcggcat tcatccatcc attcggtaac gcagatcgga tgattacggt ccttgcggta
     3061 aatccggcat gtacaggatt cattgtcctg ctcaaagtcc atgccatcaa actgctggtt
     3121 ttcattgatg atgcgggacc agccatcaac gcccaccacc ggaacgatgc cattctgctt
     3181 atcaggaaag gcgtaaattt ctttcgtcca cggattaagg ccgtactggt tggcaacgat
     3241 cagtaatgcg atgaactgcg catcgctggc atcaccttta aatgccgtct ggcgaagagt
     3301 ggtgatcagt tcctgtgggt cgacagaatc catgccgaca cgttcagcca gcttcccagc
     3361 cagcgttgcg agtgcagtac tcattcgttt tatacctctg aatcaatatc aacctggtgg
     3421 tgagcaatgg tttcaaccat gtaccggatg tgttctgcca tgcgctcctg aaactcaaca
     3481 tcgtcatcaa acgcacgggt aatggatttt ttgctggccc cgtggcgttg caaatgatcg
     3541 atgcatagcg attcaaacag gtgctggggc aggccttttt ccatgtcgtc tgccagttct
     3601 gcctctttct cttcacgggc gagctgctgg tagtgacgcg cccagctctg agcctcaaga
     3661 cgatcctgaa tgtaataagc gttcatggct gaactcctga aatagctgtg aaaatatcgc
     3721 ccgcgaaatg ccgggctgat taggaaaaca ggaaaggggg ttagtgaatg cttttgcttg
     3781 atctcagttt cagtattaat atccattttt tataacctcc ttagagctcg aattcccaaa
     3841 aaaacgggta tggagaaaca gtagagagtt gcgataaaaa gcgtcaggta ggatccgcta
     3901 atcttatgga taaaaatgct atggcatagc aaagtgtgac gccgtgcaaa taatcaatgt
     3961 ggacttttct gccgtgatta tagacacttt tgttacgcgt ttttgtcatg gctttggtcc
     4021 cgctttgtta cagaatgctt ttaataagcg gggttaccgg tttggttagc gagaagagcc
     4081 agtaaaagac gcagtgacgg caatgtctga tgcaatatgg acaattggtt tcttctctga
     4141 atggcgggag tatgaaaagt atggctgaag cgcaaaatga tcccctgctg ccgggatact
     4201 cgtttaatgc ccatctggtg gcgggtttaa cgccgattga ggccaacggt tatctcgatt
     4261 tttttatcga ccgaccgctg ggaatgaaag gttatattct caatctcacc attcgcggtc
     4321 agggggtggt gaaaaatcag ggacgagaat ttgtttgccg accgggtgat attttgctgt
     4381 tcccgccagg agagattcat cactacggtc gtcatccgga ggctcgcgaa tggtatcacc
     4441 agtgggttta ctttcgtccg cgcgcctact ggcatgaatg gcttaactgg ccgtcaatat
     4501 ttgccaatac ggggttcttt cgcccggatg aagcgcacca gccgcatttc agcgacctgt
     4561 ttgggcaaat cattaacgcc gggcaagggg aagggcgcta ttcggagctg ctggcgataa
     4621 atctgcttga gcaattgtta ctgcggcgca tggaagcgat taacgagtcg ctccatccac
     4681 cgatggataa tcgggtacgc gaggcttgtc agtacatcag cgatcacctg gcagacagca
     4741 attttgatat cgccagcgtc gcacagcatg tttgcttgtc gccgtcgcgt ctgtcacatc
     4801 ttttccgcca gcagttaggg attagcgtct taagctggcg cgaggaccaa cgtatcagcc
     4861 aggcgaagct gcttttgagc accacccgga tgcctatcgc caccgtcggt cgcaatgttg
     4921 gttttgacga tcaactctac ttctcgcggg tatttaaaaa atgcaccggg gccagcccga
     4981 gcgagttccg tgccggttgt gaagaaaaag tgaatgatgt agccgtcaag ttgtcataat
     5041 aaatcgatgc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5101 tctaaataca ttcaaatatg tatccgctca tgagacaata accctggtag cgaccgagtg
     5161 agctagctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5221 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagggaa gcggtgatcg
     5281 ccgaagtatc gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga
     5341 cgttgctggc cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagtg
     5401 atattgattt gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga
     5461 tcaacgacct tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag
     5521 aagtcaccat tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac
     5581 tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga
     5641 tcgacattga tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag
     5701 gtccagcggc ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa
     5761 atgaaacctt aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag
     5821 tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg
     5881 tcgctgccga ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag
     5941 ctagacaggc ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg
     6001 aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt agtcggcaaa taatgtcctg
     6061 tcagcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6121 aaaaggatct aggtgaagat ccgcctgcag tctagactcg agtaaggatc cagttcaccg
     6181 acaaacaaca gataaaacga aaggcccagt ctttcgactg agcctttcgt tttatttgat
     6241 gcctggcagt tccctactct cgcatgggga gaccccacac taccatcggc gctacggcgt
     6301 ttcacttctg agttcggcat ggggtcaggt gggaccaccg cgctactgcc gccaggcaaa
     6361 ttctgtttta tcagaccgct tctgcgttct gatttaatct gtatcaggct gaaaatcttc
     6421 tctcatccgc caaaacagcc aagctggaga ccgtttaaac tcaatgatga tgatgatgat
     6481 ggtcgacggc gctattcaga tcctcttctg agatgagttt ttgttcgggc ccaagcttca
     6541 aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc ttattttaac
     6601 ttgctatttc tagctctaaa acccttcgat cagatgaaaa atgtgctcag tatctctatc
     6661 actgataggg atgtcaatct ctatcactga tagggaagaa ttccagaaat catccttagc
     6721 gaaagctgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6781 agaccccgtt gatgataccg ctgccttact gggtgcatta gccagtctga atgacctgtc
     6841 acgggataat ccgaagtggt cagactggaa aatcagaggg caggaactgc tgaacagcaa
     6901 aaagtcagat agcaccacat agcagacc
//
