LOCUS       sequence-181329-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains arabinose-induced lambda Red and a tet-inducible gRNA 
            targeting arsB..
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-arsB
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(6..884)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        911..1195
                     /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     1118..1137
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             1222..1230
                     /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             1239..1655
                     /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             1661..2446
                     /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             2443..3123
                     /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      3124..3368
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(3491..4441)
                     /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      4489..4711
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     protein_bind    5277..5295
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5302..5320
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        5351..5426
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             5447..5476
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5492..5509
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5565..5582)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5735..5781
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5920..6711)
                     /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(6712..6803)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atttattatg acaacttgac ggctacatca ttcacttttt cttcacaacc ggcacggaac
       61 tcgctcgggc tggccccggt gcatttttta aatacccgcg agaagtagag ttgatcgtca
      121 aaaccaacat tgcgaccgac ggtggcgata ggcatccggg tggtgctcaa aagcagcttc
      181 gcctggctga tacgttggtc ctcgcgccag cttaagacgc taatccctaa ctgctggcgg
      241 aaaagatgtg acagacgcga cggcgacaag caaacatgct gtgcgacgct ggcgatatca
      301 aaattgctgt ctgccaggtg atcgctgatg tactgacaag cctcgcgtac ccgattatcc
      361 atcggtggat ggagcgactc gttaatcgct tccatgcgcc gcagtaacaa ttgctcaagc
      421 agatttatcg ccagcagctc cgaatagcgc ccttcccctt gcccggcgtt aatgatttgc
      481 ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg ggcgaaagaa ccccgtattg
      541 gcaaatattg acggccagtt aagccattca tgccagtagg cgcgcggacg aaagtaaacc
      601 cactggtgat accattcgcg agcctccgga tgacgaccgt agtgatgaat ctctcctggc
      661 gggaacagca aaatatcacc cggtcggcaa acaaattctc gtccctgatt tttcaccacc
      721 ccctgaccgc gaatggtgag attgagaata taacctttca ttcccagcgg tcggtcgata
      781 aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac ccgccaccag atgggcatta
      841 aacgagtatc ccggcagcag gggatcattt tgcgcttcag ccatactttt catactcccg
      901 ccattcagag aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt
      961 tactggctct tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa
     1021 agcgggacca aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa
     1081 gtccacattg attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata
     1141 agattagcgg atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg
     1201 tttttttggg aattcgagct ctaaggaggt tataaaaaat ggatattaat actgaaactg
     1261 agatcaagca aaagcattca ctaaccccct ttcctgtttt cctaatcagc ccggcatttc
     1321 gcgggcgata ttttcacagc tatttcagga gttcagccat gaacgcttat tacattcagg
     1381 atcgtcttga ggctcagagc tgggcgcgtc actaccagca gctcgcccgt gaagagaaag
     1441 aggcagaact ggcagacgac atggaaaaag gcctgcccca gcacctgttt gaatcgctat
     1501 gcatcgatca tttgcaacgc cacggggcca gcaaaaaatc cattacccgt gcgtttgatg
     1561 acgatgttga gtttcaggag cgcatggcag aacacatccg gtacatggtt gaaaccattg
     1621 ctcaccacca ggttgatatt gattcagagg tataaaacga atgagtactg cactcgcaac
     1681 gctggctggg aagctggctg aacgtgtcgg catggattct gtcgacccac aggaactgat
     1741 caccactctt cgccagacgg catttaaagg tgatgccagc gatgcgcagt tcatcgcatt
     1801 actgatcgtt gccaaccagt acggccttaa tccgtggacg aaagaaattt acgcctttcc
     1861 tgataagcag aatggcatcg ttccggtggt gggcgttgat ggctggtccc gcatcatcaa
     1921 tgaaaaccag cagtttgatg gcatggactt tgagcaggac aatgaatcct gtacatgccg
     1981 gatttaccgc aaggaccgta atcatccgat ctgcgttacc gaatggatgg atgaatgccg
     2041 ccgcgaacca ttcaaaactc gcgaaggcag agaaatcacg gggccgtggc agtcgcatcc
     2101 caaacggatg ttacgtcata aagccatgat tcagtgtgcc cgtctggcct tcggatttgc
     2161 tggtatctat gacaaggatg aagccgagcg cattgtcgaa aatactgcat acactgcaga
     2221 acgtcagccg gaacgcgaca tcactccggt taacgatgaa accatgcagg agattaacac
     2281 tctgctgatc gccctggata aaacatggga tgacgactta ttgccgctct gttcccagat
     2341 atttcgccgc gacattcgtg catcgtcaga actgacacag gccgaagcag taaaagctct
     2401 tggattcctg aaacagaaag ccgcagagca gaaggtggca gcatgacacc ggacattatc
     2461 ctgcagcgta ccgggatcga tgtgagagct gtcgaacagg gggatgatgc gtggcacaaa
     2521 ttacggctcg gcgtcatcac cgcttcagaa gttcacaacg tgatagcaaa accccgctcc
     2581 ggaaagaagt ggcctgacat gaaaatgtcc tacttccaca ccctgcttgc tgaggtttgc
     2641 accggtgtgg ctccggaagt taacgctaaa gcactggcct ggggaaaaca gtacgagaac
     2701 gacgccagaa ccctgtttga attcacttcc ggcgtgaatg ttactgaatc cccgatcatc
     2761 tatcgcgacg aaagtatgcg taccgcctgc tctcccgatg gtttatgcag tgacggcaac
     2821 ggccttgaac tgaaatgccc gtttacctcc cgggatttca tgaagttccg gctcggtggt
     2881 ttcgaggcca taaagtcagc ttacatggcc caggtgcagt acagcatgtg ggtgacgcga
     2941 aaaaatgcct ggtactttgc caactatgac ccgcgtatga agcgtgaagg cctgcattat
     3001 gtcgtgattg agcgggatga aaagtacatg gcgagttttg acgagatcgt gccggagttc
     3061 atcgaaaaaa tggacgaggc actggctgaa attggttttg tatttgggga gcaatggcga
     3121 tgacgcatcc tcacgataat atccgggtag gcgcaatcac tttcgtctac tccgttacaa
     3181 agcgaggctg ggtatttccc ggcctttctg ttatccgaaa tccactgaaa gcacagcggc
     3241 tggctgagga gataaataat aaacgagggg ctgtatgcac aaagcatctt ctgttgagtt
     3301 aagaacgagt atcgagatgg cacatagcct tgctcaaatt ggaatcaggt ttgtgccaat
     3361 accagtagaa acagacgaag aatccatggg tatggacagt tttccctttg atatgtaacg
     3421 gtgaacagtt gttctacttt tgtttgttag tcttgatgct tcactgatag atacaagagc
     3481 cataagaacc tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg gttcgttgtt
     3541 tttgcgtgag ccatgagaac gaaccattga gatcatactt actttgcatg tcactcaaaa
     3601 attttgcctc aaaactggtg agctgaattt ttgcagttaa agcatcgtgt agtgtttttc
     3661 ttagtccgtt atgtaggtag gaatctgatg taatggttgt tggtattttg tcaccattca
     3721 tttttatctg gttgttctca agttcggtta cgagatccat ttgtctatct agttcaactt
     3781 ggaaaatcaa cgtatcagtc gggcggcctc gcttatcaac caccaatttc atattgctgt
     3841 aagtgtttaa atctttactt attggtttca aaacccattg gttaagcctt ttaaactcat
     3901 ggtagttatt ttcaagcatt aacatgaact taaattcatc aaggctaatc tctatatttg
     3961 ccttgtgagt tttcttttgt gttagttctt ttaataacca ctcataaatc ctcatagagt
     4021 atttgttttc aaaagactta acatgttcca gattatattt tatgaatttt tttaactgga
     4081 aaagataagg caatatctct tcactaaaaa ctaattctaa tttttcgctt gagaacttgg
     4141 catagtttgt ccactggaaa atctcaaagc ctttaaccaa aggattcctg atttccacag
     4201 ttctcgtcat cagctctctg gttgctttag ctaatacacc ataagcattt tccctactga
     4261 tgttcatcat ctgaacgtat tggttataag tgaacgatac cgtccgttct ttccttgtag
     4321 ggttttcaat cgtggggttg agtagtgcca cacagcataa aattagcttg gtttcatgct
     4381 ccgttaagtc atagcgacta atcgctagtt catttgcttt gaaaacaact aattcagaca
     4441 tacatctcaa ttggtctagg tgattttaat cactatacca attgagatgg gctagtcaat
     4501 gataattact agtccttttc ctttgagttg tgggtatctg taaattctgc tagacctttg
     4561 ctggaaaact tgtaaattct gctagaccct ctgtaaattc cgctagacct ttgtgtgttt
     4621 tttttgttta tattcaagtg gttataattt atagaataaa gaaagaataa aaaaagataa
     4681 aaagaataga tcccagccct gtgtataact cactacttta gtcagttccg cagtattaca
     4741 aaaggatgtc gcaaacgctg tttgctcctc tacaaaacag accttaaaac cctaaaggct
     4801 taagtagcac cctcgcaagc tcggttgcgg ccgcaatcgg gcaaatcgct gaatattcct
     4861 tttgtctccg accatcaggc acctgagtcg ctgtcttttt cgtgacattc agttcgctgc
     4921 gctcacggct ctggcagtga atgggggtaa atggcactac aggcgccttt tatggattca
     4981 tgcaaggaaa ctacccataa tacaagaaaa gcccgtcacg ggcttctcag ggcgttttat
     5041 ggcgggtctg ctatgtggtg ctatctgact ttttgctgtt cagcagttcc tgccctctga
     5101 ttttccagtc tgaccacttc ggattatccc gtgacaggtc attcagactg gctaatgcac
     5161 ccagtaaggc agcggtatca tcaacggggt ctgacgctca gtggaacgaa aactcacgtt
     5221 aagggatttt ggtcatgaga ttatcagctt tcgctaagga tgatttctgg aattcttccc
     5281 tatcagtgat agagattgac atccctatca gtgatagaga tactgagcac gttacgggcg
     5341 tggtccatcc gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     5401 ttgaaaaagt ggcaccgagt cggtgctttt tttgaagctt gggcccgaac aaaaactcat
     5461 ctcagaagag gatctgaata gcgccgtcga ccatcatcat catcatcatt gagtttaaac
     5521 ggtctccagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa
     5581 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc
     5641 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
     5701 tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
     5761 agactgggcc tttcgtttta tctgttgttt gtcggtgaac tggatcctta ctcgagtcta
     5821 gactgcaggc ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5881 taaagtatat atgagtaaac ttggtctgct gacaggacat tatttgccga ctaccttggt
     5941 gatctcgcct ttcacgtagt ggacaaattc ttccaactga tctgcgcgcg aggccaagcg
     6001 atcttcttct tgtccaagat aagcctgtct agcttcaagt atgacgggct gatactgggc
     6061 cggcaggcgc tccattgccc agtcggcagc gacatccttc ggcgcgattt tgccggttac
     6121 tgcgctgtac caaatgcggg acaacgtaag cactacattt cgctcatcgc cagcccagtc
     6181 gggcggcgag ttccatagcg ttaaggtttc atttagcgcc tcaaatagat cctgttcagg
     6241 aaccggatca aagagttcct ccgccgctgg acctaccaag gcaacgctat gttctcttgc
     6301 ttttgtcagc aagatagcca gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag
     6361 aatgtcattg cgctgccatt ctccaaattg cagttcgcgc ttagctggat aacgccacgg
     6421 aatgatgtcg tcgtgcacaa caatggtgac ttctacagcg cggagaatct cgctctctcc
     6481 aggggaagcc gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag
     6541 ccttacggtc accgtaacca gcaaatcaat atcactgtgt ggcttcaggc cgccatccac
     6601 tgcggagccg tacaaatgta cggccagcaa cgtcggttcg agatggcgct cgatgacgcc
     6661 aactacctct gatagttgag tcgatacttc ggcgatcacc gcttccctca tactcttcct
     6721 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6781 atgtatttag aaaaataaac aaatagctag ctcactcggt cgctaccagg gttattgtct
     6841 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6901 atttccccga aaagtgccac ctgcatcg
//
