LOCUS       sequence-201608-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains arabinose-induced lambda Red and a tet-inducible gRNA 
            targeting adhE..
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-adhE
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    230..248
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    255..273
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        304..379
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             400..429
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             445..462
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(518..535)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      688..734
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(873..1664)
                     /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(1665..1756)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(1887..2765)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        2792..3076
                     /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     2999..3018
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             3103..3111
                     /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             3120..3536
                     /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             3542..4327
                     /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             4324..5004
                     /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      5005..5249
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(5372..6322)
                     /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      6370..6592
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 ctgctatgtg gtgctatctg actttttgct gttcagcagt tcctgccctc tgattttcca
       61 gtctgaccac ttcggattat cccgtgacag gtcattcaga ctggctaatg cacccagtaa
      121 ggcagcggta tcatcaacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
      181 tttggtcatg agattatcag ctttcgctaa ggatgatttc tggaattctt ccctatcagt
      241 gatagagatt gacatcccta tcagtgatag agatactgag cacccgaaag cacacaggga
      301 cttgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
      361 agtggcaccg agtcggtgct ttttttgaag cttgggcccg aacaaaaact catctcagaa
      421 gaggatctga atagcgccgt cgaccatcat catcatcatc attgagttta aacggtctcc
      481 agcttggctg ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac
      541 gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg
      601 accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc
      661 atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg
      721 gcctttcgtt ttatctgttg tttgtcggtg aactggatcc ttactcgagt ctagactgca
      781 ggcggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
      841 tatatgagta aacttggtct gctgacagga cattatttgc cgactacctt ggtgatctcg
      901 cctttcacgt agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct
      961 tcttgtccaa gataagcctg tctagcttca agtatgacgg gctgatactg ggccggcagg
     1021 cgctccattg cccagtcggc agcgacatcc ttcggcgcga ttttgccggt tactgcgctg
     1081 taccaaatgc gggacaacgt aagcactaca tttcgctcat cgccagccca gtcgggcggc
     1141 gagttccata gcgttaaggt ttcatttagc gcctcaaata gatcctgttc aggaaccgga
     1201 tcaaagagtt cctccgccgc tggacctacc aaggcaacgc tatgttctct tgcttttgtc
     1261 agcaagatag ccagatcaat gtcgatcgtg gctggctcga agatacctgc aagaatgtca
     1321 ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg gataacgcca cggaatgatg
     1381 tcgtcgtgca caacaatggt gacttctaca gcgcggagaa tctcgctctc tccaggggaa
     1441 gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc aagccttacg
     1501 gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca ggccgccatc cactgcggag
     1561 ccgtacaaat gtacggccag caacgtcggt tcgagatggc gctcgatgac gccaactacc
     1621 tctgatagtt gagtcgatac ttcggcgatc accgcttccc tcatactctt cctttttcaa
     1681 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     1741 tagaaaaata aacaaatagc tagctcactc ggtcgctacc agggttattg tctcatgagc
     1801 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     1861 cgaaaagtgc cacctgcatc gatttattat gacaacttga cggctacatc attcactttt
     1921 tcttcacaac cggcacggaa ctcgctcggg ctggccccgg tgcatttttt aaatacccgc
     1981 gagaagtaga gttgatcgtc aaaaccaaca ttgcgaccga cggtggcgat aggcatccgg
     2041 gtggtgctca aaagcagctt cgcctggctg atacgttggt cctcgcgcca gcttaagacg
     2101 ctaatcccta actgctggcg gaaaagatgt gacagacgcg acggcgacaa gcaaacatgc
     2161 tgtgcgacgc tggcgatatc aaaattgctg tctgccaggt gatcgctgat gtactgacaa
     2221 gcctcgcgta cccgattatc catcggtgga tggagcgact cgttaatcgc ttccatgcgc
     2281 cgcagtaaca attgctcaag cagatttatc gccagcagct ccgaatagcg cccttcccct
     2341 tgcccggcgt taatgatttg cccaaacagg tcgctgaaat gcggctggtg cgcttcatcc
     2401 gggcgaaaga accccgtatt ggcaaatatt gacggccagt taagccattc atgccagtag
     2461 gcgcgcggac gaaagtaaac ccactggtga taccattcgc gagcctccgg atgacgaccg
     2521 tagtgatgaa tctctcctgg cgggaacagc aaaatatcac ccggtcggca aacaaattct
     2581 cgtccctgat ttttcaccac cccctgaccg cgaatggtga gattgagaat ataacctttc
     2641 attcccagcg gtcggtcgat aaaaaaatcg agataaccgt tggcctcaat cggcgttaaa
     2701 cccgccacca gatgggcatt aaacgagtat cccggcagca ggggatcatt ttgcgcttca
     2761 gccatacttt tcatactccc gccattcaga gaagaaacca attgtccata ttgcatcaga
     2821 cattgccgtc actgcgtctt ttactggctc ttctcgctaa ccaaaccggt aaccccgctt
     2881 attaaaagca ttctgtaaca aagcgggacc aaagccatga caaaaacgcg taacaaaagt
     2941 gtctataatc acggcagaaa agtccacatt gattatttgc acggcgtcac actttgctat
     3001 gccatagcat ttttatccat aagattagcg gatcctacct gacgcttttt atcgcaactc
     3061 tctactgttt ctccataccc gtttttttgg gaattcgagc tctaaggagg ttataaaaaa
     3121 tggatattaa tactgaaact gagatcaagc aaaagcattc actaaccccc tttcctgttt
     3181 tcctaatcag cccggcattt cgcgggcgat attttcacag ctatttcagg agttcagcca
     3241 tgaacgctta ttacattcag gatcgtcttg aggctcagag ctgggcgcgt cactaccagc
     3301 agctcgcccg tgaagagaaa gaggcagaac tggcagacga catggaaaaa ggcctgcccc
     3361 agcacctgtt tgaatcgcta tgcatcgatc atttgcaacg ccacggggcc agcaaaaaat
     3421 ccattacccg tgcgtttgat gacgatgttg agtttcagga gcgcatggca gaacacatcc
     3481 ggtacatggt tgaaaccatt gctcaccacc aggttgatat tgattcagag gtataaaacg
     3541 aatgagtact gcactcgcaa cgctggctgg gaagctggct gaacgtgtcg gcatggattc
     3601 tgtcgaccca caggaactga tcaccactct tcgccagacg gcatttaaag gtgatgccag
     3661 cgatgcgcag ttcatcgcat tactgatcgt tgccaaccag tacggcctta atccgtggac
     3721 gaaagaaatt tacgcctttc ctgataagca gaatggcatc gttccggtgg tgggcgttga
     3781 tggctggtcc cgcatcatca atgaaaacca gcagtttgat ggcatggact ttgagcagga
     3841 caatgaatcc tgtacatgcc ggatttaccg caaggaccgt aatcatccga tctgcgttac
     3901 cgaatggatg gatgaatgcc gccgcgaacc attcaaaact cgcgaaggca gagaaatcac
     3961 ggggccgtgg cagtcgcatc ccaaacggat gttacgtcat aaagccatga ttcagtgtgc
     4021 ccgtctggcc ttcggatttg ctggtatcta tgacaaggat gaagccgagc gcattgtcga
     4081 aaatactgca tacactgcag aacgtcagcc ggaacgcgac atcactccgg ttaacgatga
     4141 aaccatgcag gagattaaca ctctgctgat cgccctggat aaaacatggg atgacgactt
     4201 attgccgctc tgttcccaga tatttcgccg cgacattcgt gcatcgtcag aactgacaca
     4261 ggccgaagca gtaaaagctc ttggattcct gaaacagaaa gccgcagagc agaaggtggc
     4321 agcatgacac cggacattat cctgcagcgt accgggatcg atgtgagagc tgtcgaacag
     4381 ggggatgatg cgtggcacaa attacggctc ggcgtcatca ccgcttcaga agttcacaac
     4441 gtgatagcaa aaccccgctc cggaaagaag tggcctgaca tgaaaatgtc ctacttccac
     4501 accctgcttg ctgaggtttg caccggtgtg gctccggaag ttaacgctaa agcactggcc
     4561 tggggaaaac agtacgagaa cgacgccaga accctgtttg aattcacttc cggcgtgaat
     4621 gttactgaat ccccgatcat ctatcgcgac gaaagtatgc gtaccgcctg ctctcccgat
     4681 ggtttatgca gtgacggcaa cggccttgaa ctgaaatgcc cgtttacctc ccgggatttc
     4741 atgaagttcc ggctcggtgg tttcgaggcc ataaagtcag cttacatggc ccaggtgcag
     4801 tacagcatgt gggtgacgcg aaaaaatgcc tggtactttg ccaactatga cccgcgtatg
     4861 aagcgtgaag gcctgcatta tgtcgtgatt gagcgggatg aaaagtacat ggcgagtttt
     4921 gacgagatcg tgccggagtt catcgaaaaa atggacgagg cactggctga aattggtttt
     4981 gtatttgggg agcaatggcg atgacgcatc ctcacgataa tatccgggta ggcgcaatca
     5041 ctttcgtcta ctccgttaca aagcgaggct gggtatttcc cggcctttct gttatccgaa
     5101 atccactgaa agcacagcgg ctggctgagg agataaataa taaacgaggg gctgtatgca
     5161 caaagcatct tctgttgagt taagaacgag tatcgagatg gcacatagcc ttgctcaaat
     5221 tggaatcagg tttgtgccaa taccagtaga aacagacgaa gaatccatgg gtatggacag
     5281 ttttcccttt gatatgtaac ggtgaacagt tgttctactt ttgtttgtta gtcttgatgc
     5341 ttcactgata gatacaagag ccataagaac ctcagatcct tccgtattta gccagtatgt
     5401 tctctagtgt ggttcgttgt ttttgcgtga gccatgagaa cgaaccattg agatcatact
     5461 tactttgcat gtcactcaaa aattttgcct caaaactggt gagctgaatt tttgcagtta
     5521 aagcatcgtg tagtgttttt cttagtccgt tatgtaggta ggaatctgat gtaatggttg
     5581 ttggtatttt gtcaccattc atttttatct ggttgttctc aagttcggtt acgagatcca
     5641 tttgtctatc tagttcaact tggaaaatca acgtatcagt cgggcggcct cgcttatcaa
     5701 ccaccaattt catattgctg taagtgttta aatctttact tattggtttc aaaacccatt
     5761 ggttaagcct tttaaactca tggtagttat tttcaagcat taacatgaac ttaaattcat
     5821 caaggctaat ctctatattt gccttgtgag ttttcttttg tgttagttct tttaataacc
     5881 actcataaat cctcatagag tatttgtttt caaaagactt aacatgttcc agattatatt
     5941 ttatgaattt ttttaactgg aaaagataag gcaatatctc ttcactaaaa actaattcta
     6001 atttttcgct tgagaacttg gcatagtttg tccactggaa aatctcaaag cctttaacca
     6061 aaggattcct gatttccaca gttctcgtca tcagctctct ggttgcttta gctaatacac
     6121 cataagcatt ttccctactg atgttcatca tctgaacgta ttggttataa gtgaacgata
     6181 ccgtccgttc tttccttgta gggttttcaa tcgtggggtt gagtagtgcc acacagcata
     6241 aaattagctt ggtttcatgc tccgttaagt catagcgact aatcgctagt tcatttgctt
     6301 tgaaaacaac taattcagac atacatctca attggtctag gtgattttaa tcactatacc
     6361 aattgagatg ggctagtcaa tgataattac tagtcctttt cctttgagtt gtgggtatct
     6421 gtaaattctg ctagaccttt gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt
     6481 ccgctagacc tttgtgtgtt ttttttgttt atattcaagt ggttataatt tatagaataa
     6541 agaaagaata aaaaaagata aaaagaatag atcccagccc tgtgtataac tcactacttt
     6601 agtcagttcc gcagtattac aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca
     6661 gaccttaaaa ccctaaaggc ttaagtagca ccctcgcaag ctcggttgcg gccgcaatcg
     6721 ggcaaatcgc tgaatattcc ttttgtctcc gaccatcagg cacctgagtc gctgtctttt
     6781 tcgtgacatt cagttcgctg cgctcacggc tctggcagtg aatgggggta aatggcacta
     6841 caggcgcctt ttatggattc atgcaaggaa actacccata atacaagaaa agcccgtcac
     6901 gggcttctca gggcgtttta tggcgggt
//
