LOCUS       sequence-311764-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  aTc inducible gRNA targeting pheT along with arabinose-inducible 
            lambda red enzymes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6928)
  AUTHORS   Moreb EA, Hutmacher M, Lynch MD
  TITLE     CRISPR-Cas ""Non-Target"" Sites Inhibit On-Target Cutting Rates.
  JOURNAL   CRISPR J. 2020 Dec;3(6):550-561. doi: 10.1089/crispr.2020.0065.
  PUBMED    33346713
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"; doi: 
            "10.1089/crispr.2020"; journalName: "CRISPR J"; date: "2020-12"; 
            volume: "3"; issue: "6"; pages: "550-561"
COMMENT     Sequence Label: pKDsgRNA-pheT
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(11..889)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        916..1200
                     /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     1123..1142
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             1227..1235
                     /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             1244..1660
                     /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             1666..2451
                     /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             2448..3128
                     /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      3129..3373
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(3496..4446)
                     /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      4494..4716
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     protein_bind    5282..5300
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5307..5325
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        5356..5431
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             5452..5481
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5497..5514
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5570..5587)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5740..5786
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5925..6716)
                     /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(6717..6808)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catcgattta ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac
       61 ggaactcgct cgggctggcc ccggtgcatt ttttaaatac ccgcgagaag tagagttgat
      121 cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca
      181 gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct
      241 ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga
      301 tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat
      361 tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct
      421 caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga
      481 tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg
      541 tattggcaaa tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt
      601 aaacccactg gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc
      661 ctggcgggaa cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca
      721 ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt
      781 cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg
      841 cattaaacga gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac
      901 tcccgccatt cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg
      961 tcttttactg gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt
     1021 aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca
     1081 gaaaagtcca cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat
     1141 ccataagatt agcggatcct acctgacgct ttttatcgca actctctact gtttctccat
     1201 acccgttttt ttgggaattc gagctctaag gaggttataa aaaatggata ttaatactga
     1261 aactgagatc aagcaaaagc attcactaac cccctttcct gttttcctaa tcagcccggc
     1321 atttcgcggg cgatattttc acagctattt caggagttca gccatgaacg cttattacat
     1381 tcaggatcgt cttgaggctc agagctgggc gcgtcactac cagcagctcg cccgtgaaga
     1441 gaaagaggca gaactggcag acgacatgga aaaaggcctg ccccagcacc tgtttgaatc
     1501 gctatgcatc gatcatttgc aacgccacgg ggccagcaaa aaatccatta cccgtgcgtt
     1561 tgatgacgat gttgagtttc aggagcgcat ggcagaacac atccggtaca tggttgaaac
     1621 cattgctcac caccaggttg atattgattc agaggtataa aacgaatgag tactgcactc
     1681 gcaacgctgg ctgggaagct ggctgaacgt gtcggcatgg attctgtcga cccacaggaa
     1741 ctgatcacca ctcttcgcca gacggcattt aaaggtgatg ccagcgatgc gcagttcatc
     1801 gcattactga tcgttgccaa ccagtacggc cttaatccgt ggacgaaaga aatttacgcc
     1861 tttcctgata agcagaatgg catcgttccg gtggtgggcg ttgatggctg gtcccgcatc
     1921 atcaatgaaa accagcagtt tgatggcatg gactttgagc aggacaatga atcctgtaca
     1981 tgccggattt accgcaagga ccgtaatcat ccgatctgcg ttaccgaatg gatggatgaa
     2041 tgccgccgcg aaccattcaa aactcgcgaa ggcagagaaa tcacggggcc gtggcagtcg
     2101 catcccaaac ggatgttacg tcataaagcc atgattcagt gtgcccgtct ggccttcgga
     2161 tttgctggta tctatgacaa ggatgaagcc gagcgcattg tcgaaaatac tgcatacact
     2221 gcagaacgtc agccggaacg cgacatcact ccggttaacg atgaaaccat gcaggagatt
     2281 aacactctgc tgatcgccct ggataaaaca tgggatgacg acttattgcc gctctgttcc
     2341 cagatatttc gccgcgacat tcgtgcatcg tcagaactga cacaggccga agcagtaaaa
     2401 gctcttggat tcctgaaaca gaaagccgca gagcagaagg tggcagcatg acaccggaca
     2461 ttatcctgca gcgtaccggg atcgatgtga gagctgtcga acagggggat gatgcgtggc
     2521 acaaattacg gctcggcgtc atcaccgctt cagaagttca caacgtgata gcaaaacccc
     2581 gctccggaaa gaagtggcct gacatgaaaa tgtcctactt ccacaccctg cttgctgagg
     2641 tttgcaccgg tgtggctccg gaagttaacg ctaaagcact ggcctgggga aaacagtacg
     2701 agaacgacgc cagaaccctg tttgaattca cttccggcgt gaatgttact gaatccccga
     2761 tcatctatcg cgacgaaagt atgcgtaccg cctgctctcc cgatggttta tgcagtgacg
     2821 gcaacggcct tgaactgaaa tgcccgttta cctcccggga tttcatgaag ttccggctcg
     2881 gtggtttcga ggccataaag tcagcttaca tggcccaggt gcagtacagc atgtgggtga
     2941 cgcgaaaaaa tgcctggtac tttgccaact atgacccgcg tatgaagcgt gaaggcctgc
     3001 attatgtcgt gattgagcgg gatgaaaagt acatggcgag ttttgacgag atcgtgccgg
     3061 agttcatcga aaaaatggac gaggcactgg ctgaaattgg ttttgtattt ggggagcaat
     3121 ggcgatgacg catcctcacg ataatatccg ggtaggcgca atcactttcg tctactccgt
     3181 tacaaagcga ggctgggtat ttcccggcct ttctgttatc cgaaatccac tgaaagcaca
     3241 gcggctggct gaggagataa ataataaacg aggggctgta tgcacaaagc atcttctgtt
     3301 gagttaagaa cgagtatcga gatggcacat agccttgctc aaattggaat caggtttgtg
     3361 ccaataccag tagaaacaga cgaagaatcc atgggtatgg acagttttcc ctttgatatg
     3421 taacggtgaa cagttgttct acttttgttt gttagtcttg atgcttcact gatagataca
     3481 agagccataa gaacctcaga tccttccgta tttagccagt atgttctcta gtgtggttcg
     3541 ttgtttttgc gtgagccatg agaacgaacc attgagatca tacttacttt gcatgtcact
     3601 caaaaatttt gcctcaaaac tggtgagctg aatttttgca gttaaagcat cgtgtagtgt
     3661 ttttcttagt ccgttatgta ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc
     3721 attcattttt atctggttgt tctcaagttc ggttacgaga tccatttgtc tatctagttc
     3781 aacttggaaa atcaacgtat cagtcgggcg gcctcgctta tcaaccacca atttcatatt
     3841 gctgtaagtg tttaaatctt tacttattgg tttcaaaacc cattggttaa gccttttaaa
     3901 ctcatggtag ttattttcaa gcattaacat gaacttaaat tcatcaaggc taatctctat
     3961 atttgccttg tgagttttct tttgtgttag ttcttttaat aaccactcat aaatcctcat
     4021 agagtatttg ttttcaaaag acttaacatg ttccagatta tattttatga atttttttaa
     4081 ctggaaaaga taaggcaata tctcttcact aaaaactaat tctaattttt cgcttgagaa
     4141 cttggcatag tttgtccact ggaaaatctc aaagccttta accaaaggat tcctgatttc
     4201 cacagttctc gtcatcagct ctctggttgc tttagctaat acaccataag cattttccct
     4261 actgatgttc atcatctgaa cgtattggtt ataagtgaac gataccgtcc gttctttcct
     4321 tgtagggttt tcaatcgtgg ggttgagtag tgccacacag cataaaatta gcttggtttc
     4381 atgctccgtt aagtcatagc gactaatcgc tagttcattt gctttgaaaa caactaattc
     4441 agacatacat ctcaattggt ctaggtgatt ttaatcacta taccaattga gatgggctag
     4501 tcaatgataa ttactagtcc ttttcctttg agttgtgggt atctgtaaat tctgctagac
     4561 ctttgctgga aaacttgtaa attctgctag accctctgta aattccgcta gacctttgtg
     4621 tgtttttttt gtttatattc aagtggttat aatttataga ataaagaaag aataaaaaaa
     4681 gataaaaaga atagatccca gccctgtgta taactcacta ctttagtcag ttccgcagta
     4741 ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt aaaaccctaa
     4801 aggcttaagt agcaccctcg caagctcggt tgcggccgca atcgggcaaa tcgctgaata
     4861 ttccttttgt ctccgaccat caggcacctg agtcgctgtc tttttcgtga cattcagttc
     4921 gctgcgctca cggctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg
     4981 attcatgcaa ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt
     5041 tttatggcgg gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc
     5101 tctgattttc cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa
     5161 tgcacccagt aaggcagcgg tatcatcaac ggggtctgac gctcagtgga acgaaaactc
     5221 acgttaaggg attttggtca tgagattatc agctttcgct aaggatgatt tctggaattc
     5281 ttccctatca gtgatagaga ttgacatccc tatcagtgat agagatactg agcaccccgc
     5341 gttatcttgg ccgtggtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     5401 tcaacttgaa aaagtggcac cgagtcggtg ctttttttga agcttgggcc cgaacaaaaa
     5461 ctcatctcag aagaggatct gaatagcgcc gtcgaccatc atcatcatca tcattgagtt
     5521 taaacggtct ccagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga
     5581 ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg
     5641 tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg
     5701 tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag
     5761 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaactggat ccttactcga
     5821 gtctagactg caggcggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5881 caatctaaag tatatatgag taaacttggt ctgctgacag gacattattt gccgactacc
     5941 ttggtgatct cgcctttcac gtagtggaca aattcttcca actgatctgc gcgcgaggcc
     6001 aagcgatctt cttcttgtcc aagataagcc tgtctagctt caagtatgac gggctgatac
     6061 tgggccggca ggcgctccat tgcccagtcg gcagcgacat ccttcggcgc gattttgccg
     6121 gttactgcgc tgtaccaaat gcgggacaac gtaagcacta catttcgctc atcgccagcc
     6181 cagtcgggcg gcgagttcca tagcgttaag gtttcattta gcgcctcaaa tagatcctgt
     6241 tcaggaaccg gatcaaagag ttcctccgcc gctggaccta ccaaggcaac gctatgttct
     6301 cttgcttttg tcagcaagat agccagatca atgtcgatcg tggctggctc gaagatacct
     6361 gcaagaatgt cattgcgctg ccattctcca aattgcagtt cgcgcttagc tggataacgc
     6421 cacggaatga tgtcgtcgtg cacaacaatg gtgacttcta cagcgcggag aatctcgctc
     6481 tctccagggg aagccgaagt ttccaaaagg tcgttgatca aagctcgccg cgttgtttca
     6541 tcaagcctta cggtcaccgt aaccagcaaa tcaatatcac tgtgtggctt caggccgcca
     6601 tccactgcgg agccgtacaa atgtacggcc agcaacgtcg gttcgagatg gcgctcgatg
     6661 acgccaacta cctctgatag ttgagtcgat acttcggcga tcaccgcttc cctcatactc
     6721 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6781 tttgaatgta tttagaaaaa taaacaaata gctagctcac tcggtcgcta ccagggttat
     6841 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6901 cgcacatttc cccgaaaagt gccacctg
//
