LOCUS       sequence-181335-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains arabinose-induced lambda Red and a tet-inducible gRNA 
            targeting rssA..
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-rssA
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(16..894)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        921..1205
                     /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     1128..1147
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             1232..1240
                     /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             1249..1665
                     /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             1671..2456
                     /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             2453..3133
                     /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      3134..3378
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(3501..4451)
                     /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      4499..4721
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     protein_bind    5287..5305
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5312..5330
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        5361..5436
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             5457..5486
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5502..5519
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5575..5592)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5745..5791
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5930..6721)
                     /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(6722..6813)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acctgcatcg atttattatg acaacttgac ggctacatca ttcacttttt cttcacaacc
       61 ggcacggaac tcgctcgggc tggccccggt gcatttttta aatacccgcg agaagtagag
      121 ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata ggcatccggg tggtgctcaa
      181 aagcagcttc gcctggctga tacgttggtc ctcgcgccag cttaagacgc taatccctaa
      241 ctgctggcgg aaaagatgtg acagacgcga cggcgacaag caaacatgct gtgcgacgct
      301 ggcgatatca aaattgctgt ctgccaggtg atcgctgatg tactgacaag cctcgcgtac
      361 ccgattatcc atcggtggat ggagcgactc gttaatcgct tccatgcgcc gcagtaacaa
      421 ttgctcaagc agatttatcg ccagcagctc cgaatagcgc ccttcccctt gcccggcgtt
      481 aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg ggcgaaagaa
      541 ccccgtattg gcaaatattg acggccagtt aagccattca tgccagtagg cgcgcggacg
      601 aaagtaaacc cactggtgat accattcgcg agcctccgga tgacgaccgt agtgatgaat
      661 ctctcctggc gggaacagca aaatatcacc cggtcggcaa acaaattctc gtccctgatt
      721 tttcaccacc ccctgaccgc gaatggtgag attgagaata taacctttca ttcccagcgg
      781 tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac ccgccaccag
      841 atgggcatta aacgagtatc ccggcagcag gggatcattt tgcgcttcag ccatactttt
      901 catactcccg ccattcagag aagaaaccaa ttgtccatat tgcatcagac attgccgtca
      961 ctgcgtcttt tactggctct tctcgctaac caaaccggta accccgctta ttaaaagcat
     1021 tctgtaacaa agcgggacca aagccatgac aaaaacgcgt aacaaaagtg tctataatca
     1081 cggcagaaaa gtccacattg attatttgca cggcgtcaca ctttgctatg ccatagcatt
     1141 tttatccata agattagcgg atcctacctg acgcttttta tcgcaactct ctactgtttc
     1201 tccatacccg tttttttggg aattcgagct ctaaggaggt tataaaaaat ggatattaat
     1261 actgaaactg agatcaagca aaagcattca ctaaccccct ttcctgtttt cctaatcagc
     1321 ccggcatttc gcgggcgata ttttcacagc tatttcagga gttcagccat gaacgcttat
     1381 tacattcagg atcgtcttga ggctcagagc tgggcgcgtc actaccagca gctcgcccgt
     1441 gaagagaaag aggcagaact ggcagacgac atggaaaaag gcctgcccca gcacctgttt
     1501 gaatcgctat gcatcgatca tttgcaacgc cacggggcca gcaaaaaatc cattacccgt
     1561 gcgtttgatg acgatgttga gtttcaggag cgcatggcag aacacatccg gtacatggtt
     1621 gaaaccattg ctcaccacca ggttgatatt gattcagagg tataaaacga atgagtactg
     1681 cactcgcaac gctggctggg aagctggctg aacgtgtcgg catggattct gtcgacccac
     1741 aggaactgat caccactctt cgccagacgg catttaaagg tgatgccagc gatgcgcagt
     1801 tcatcgcatt actgatcgtt gccaaccagt acggccttaa tccgtggacg aaagaaattt
     1861 acgcctttcc tgataagcag aatggcatcg ttccggtggt gggcgttgat ggctggtccc
     1921 gcatcatcaa tgaaaaccag cagtttgatg gcatggactt tgagcaggac aatgaatcct
     1981 gtacatgccg gatttaccgc aaggaccgta atcatccgat ctgcgttacc gaatggatgg
     2041 atgaatgccg ccgcgaacca ttcaaaactc gcgaaggcag agaaatcacg gggccgtggc
     2101 agtcgcatcc caaacggatg ttacgtcata aagccatgat tcagtgtgcc cgtctggcct
     2161 tcggatttgc tggtatctat gacaaggatg aagccgagcg cattgtcgaa aatactgcat
     2221 acactgcaga acgtcagccg gaacgcgaca tcactccggt taacgatgaa accatgcagg
     2281 agattaacac tctgctgatc gccctggata aaacatggga tgacgactta ttgccgctct
     2341 gttcccagat atttcgccgc gacattcgtg catcgtcaga actgacacag gccgaagcag
     2401 taaaagctct tggattcctg aaacagaaag ccgcagagca gaaggtggca gcatgacacc
     2461 ggacattatc ctgcagcgta ccgggatcga tgtgagagct gtcgaacagg gggatgatgc
     2521 gtggcacaaa ttacggctcg gcgtcatcac cgcttcagaa gttcacaacg tgatagcaaa
     2581 accccgctcc ggaaagaagt ggcctgacat gaaaatgtcc tacttccaca ccctgcttgc
     2641 tgaggtttgc accggtgtgg ctccggaagt taacgctaaa gcactggcct ggggaaaaca
     2701 gtacgagaac gacgccagaa ccctgtttga attcacttcc ggcgtgaatg ttactgaatc
     2761 cccgatcatc tatcgcgacg aaagtatgcg taccgcctgc tctcccgatg gtttatgcag
     2821 tgacggcaac ggccttgaac tgaaatgccc gtttacctcc cgggatttca tgaagttccg
     2881 gctcggtggt ttcgaggcca taaagtcagc ttacatggcc caggtgcagt acagcatgtg
     2941 ggtgacgcga aaaaatgcct ggtactttgc caactatgac ccgcgtatga agcgtgaagg
     3001 cctgcattat gtcgtgattg agcgggatga aaagtacatg gcgagttttg acgagatcgt
     3061 gccggagttc atcgaaaaaa tggacgaggc actggctgaa attggttttg tatttgggga
     3121 gcaatggcga tgacgcatcc tcacgataat atccgggtag gcgcaatcac tttcgtctac
     3181 tccgttacaa agcgaggctg ggtatttccc ggcctttctg ttatccgaaa tccactgaaa
     3241 gcacagcggc tggctgagga gataaataat aaacgagggg ctgtatgcac aaagcatctt
     3301 ctgttgagtt aagaacgagt atcgagatgg cacatagcct tgctcaaatt ggaatcaggt
     3361 ttgtgccaat accagtagaa acagacgaag aatccatggg tatggacagt tttccctttg
     3421 atatgtaacg gtgaacagtt gttctacttt tgtttgttag tcttgatgct tcactgatag
     3481 atacaagagc cataagaacc tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg
     3541 gttcgttgtt tttgcgtgag ccatgagaac gaaccattga gatcatactt actttgcatg
     3601 tcactcaaaa attttgcctc aaaactggtg agctgaattt ttgcagttaa agcatcgtgt
     3661 agtgtttttc ttagtccgtt atgtaggtag gaatctgatg taatggttgt tggtattttg
     3721 tcaccattca tttttatctg gttgttctca agttcggtta cgagatccat ttgtctatct
     3781 agttcaactt ggaaaatcaa cgtatcagtc gggcggcctc gcttatcaac caccaatttc
     3841 atattgctgt aagtgtttaa atctttactt attggtttca aaacccattg gttaagcctt
     3901 ttaaactcat ggtagttatt ttcaagcatt aacatgaact taaattcatc aaggctaatc
     3961 tctatatttg ccttgtgagt tttcttttgt gttagttctt ttaataacca ctcataaatc
     4021 ctcatagagt atttgttttc aaaagactta acatgttcca gattatattt tatgaatttt
     4081 tttaactgga aaagataagg caatatctct tcactaaaaa ctaattctaa tttttcgctt
     4141 gagaacttgg catagtttgt ccactggaaa atctcaaagc ctttaaccaa aggattcctg
     4201 atttccacag ttctcgtcat cagctctctg gttgctttag ctaatacacc ataagcattt
     4261 tccctactga tgttcatcat ctgaacgtat tggttataag tgaacgatac cgtccgttct
     4321 ttccttgtag ggttttcaat cgtggggttg agtagtgcca cacagcataa aattagcttg
     4381 gtttcatgct ccgttaagtc atagcgacta atcgctagtt catttgcttt gaaaacaact
     4441 aattcagaca tacatctcaa ttggtctagg tgattttaat cactatacca attgagatgg
     4501 gctagtcaat gataattact agtccttttc ctttgagttg tgggtatctg taaattctgc
     4561 tagacctttg ctggaaaact tgtaaattct gctagaccct ctgtaaattc cgctagacct
     4621 ttgtgtgttt tttttgttta tattcaagtg gttataattt atagaataaa gaaagaataa
     4681 aaaaagataa aaagaataga tcccagccct gtgtataact cactacttta gtcagttccg
     4741 cagtattaca aaaggatgtc gcaaacgctg tttgctcctc tacaaaacag accttaaaac
     4801 cctaaaggct taagtagcac cctcgcaagc tcggttgcgg ccgcaatcgg gcaaatcgct
     4861 gaatattcct tttgtctccg accatcaggc acctgagtcg ctgtcttttt cgtgacattc
     4921 agttcgctgc gctcacggct ctggcagtga atgggggtaa atggcactac aggcgccttt
     4981 tatggattca tgcaaggaaa ctacccataa tacaagaaaa gcccgtcacg ggcttctcag
     5041 ggcgttttat ggcgggtctg ctatgtggtg ctatctgact ttttgctgtt cagcagttcc
     5101 tgccctctga ttttccagtc tgaccacttc ggattatccc gtgacaggtc attcagactg
     5161 gctaatgcac ccagtaaggc agcggtatca tcaacggggt ctgacgctca gtggaacgaa
     5221 aactcacgtt aagggatttt ggtcatgaga ttatcagctt tcgctaagga tgatttctgg
     5281 aattcttccc tatcagtgat agagattgac atccctatca gtgatagaga tactgagcac
     5341 cagcgcggtg ggttactgcg gttttagagc tagaaatagc aagttaaaat aaggctagtc
     5401 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttgaagctt gggcccgaac
     5461 aaaaactcat ctcagaagag gatctgaata gcgccgtcga ccatcatcat catcatcatt
     5521 gagtttaaac ggtctccagc ttggctgttt tggcggatga gagaagattt tcagcctgat
     5581 acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag
     5641 cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg
     5701 tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg
     5761 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac tggatcctta
     5821 ctcgagtcta gactgcaggc ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5881 taaatcaatc taaagtatat atgagtaaac ttggtctgct gacaggacat tatttgccga
     5941 ctaccttggt gatctcgcct ttcacgtagt ggacaaattc ttccaactga tctgcgcgcg
     6001 aggccaagcg atcttcttct tgtccaagat aagcctgtct agcttcaagt atgacgggct
     6061 gatactgggc cggcaggcgc tccattgccc agtcggcagc gacatccttc ggcgcgattt
     6121 tgccggttac tgcgctgtac caaatgcggg acaacgtaag cactacattt cgctcatcgc
     6181 cagcccagtc gggcggcgag ttccatagcg ttaaggtttc atttagcgcc tcaaatagat
     6241 cctgttcagg aaccggatca aagagttcct ccgccgctgg acctaccaag gcaacgctat
     6301 gttctcttgc ttttgtcagc aagatagcca gatcaatgtc gatcgtggct ggctcgaaga
     6361 tacctgcaag aatgtcattg cgctgccatt ctccaaattg cagttcgcgc ttagctggat
     6421 aacgccacgg aatgatgtcg tcgtgcacaa caatggtgac ttctacagcg cggagaatct
     6481 cgctctctcc aggggaagcc gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg
     6541 tttcatcaag ccttacggtc accgtaacca gcaaatcaat atcactgtgt ggcttcaggc
     6601 cgccatccac tgcggagccg tacaaatgta cggccagcaa cgtcggttcg agatggcgct
     6661 cgatgacgcc aactacctct gatagttgag tcgatacttc ggcgatcacc gcttccctca
     6721 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6781 acatatttga atgtatttag aaaaataaac aaatagctag ctcactcggt cgctaccagg
     6841 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6901 ttccgcgcac atttccccga aaagtgcc
//
