LOCUS       sequence-218622-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses gRNA for Cas9 FRT targetting.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6928)
  AUTHORS   Swings T, Marciano DC, Atri B, Bosserman RE, Wang C, Leysen M, Bonte
            C, Schalck T, Furey I, Van den Bergh B, Verstraeten N, Christie PJ, 
            Herman C, Lichtarge O, Michiels J
  TITLE     CRISPR-FRT targets shared sites in a knock-out collection for 
            off-the-shelf genome editing.
  JOURNAL   Nat Commun. 2018 Jun 8;9(1):2231. doi: 10.1038/s41467-018-04651-5.
  PUBMED    29884781
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: "Nat 
            Commun."; date: "2018-06-8"; volume: "9(1):2231. doi"; pages: " 
            10.1038/s41467-018-04651-5"
COMMENT     Sequence Label: pKDsgRNA-FRT
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..285
                     /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     208..227
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             312..320
                     /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             329..745
                     /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             751..1536
                     /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             1533..2213
                     /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      2214..2458
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(2581..3531)
                     /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      3579..3801
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     protein_bind    4367..4385
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4392..4410
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        4441..4516
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             4537..4566
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4582..4599
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4655..4672)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4825..4871
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5010..5801)
                     /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(5802..5893)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(6024..6902)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct
       61 tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca
      121 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg
      181 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg
      241 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg tttttttggg
      301 aattcgagct ctaaggaggt tataaaaaat ggatattaat actgaaactg agatcaagca
      361 aaagcattca ctaaccccct ttcctgtttt cctaatcagc ccggcatttc gcgggcgata
      421 ttttcacagc tatttcagga gttcagccat gaacgcttat tacattcagg atcgtcttga
      481 ggctcagagc tgggcgcgtc actaccagca gctcgcccgt gaagagaaag aggcagaact
      541 ggcagacgac atggaaaaag gcctgcccca gcacctgttt gaatcgctat gcatcgatca
      601 tttgcaacgc cacggggcca gcaaaaaatc cattacccgt gcgtttgatg acgatgttga
      661 gtttcaggag cgcatggcag aacacatccg gtacatggtt gaaaccattg ctcaccacca
      721 ggttgatatt gattcagagg tataaaacga atgagtactg cactcgcaac gctggctggg
      781 aagctggctg aacgtgtcgg catggattct gtcgacccac aggaactgat caccactctt
      841 cgccagacgg catttaaagg tgatgccagc gatgcgcagt tcatcgcatt actgatcgtt
      901 gccaaccagt acggccttaa tccgtggacg aaagaaattt acgcctttcc tgataagcag
      961 aatggcatcg ttccggtggt gggcgttgat ggctggtccc gcatcatcaa tgaaaaccag
     1021 cagtttgatg gcatggactt tgagcaggac aatgaatcct gtacatgccg gatttaccgc
     1081 aaggaccgta atcatccgat ctgcgttacc gaatggatgg atgaatgccg ccgcgaacca
     1141 ttcaaaactc gcgaaggcag agaaatcacg gggccgtggc agtcgcatcc caaacggatg
     1201 ttacgtcata aagccatgat tcagtgtgcc cgtctggcct tcggatttgc tggtatctat
     1261 gacaaggatg aagccgagcg cattgtcgaa aatactgcat acactgcaga acgtcagccg
     1321 gaacgcgaca tcactccggt taacgatgaa accatgcagg agattaacac tctgctgatc
     1381 gccctggata aaacatggga tgacgactta ttgccgctct gttcccagat atttcgccgc
     1441 gacattcgtg catcgtcaga actgacacag gccgaagcag taaaagctct tggattcctg
     1501 aaacagaaag ccgcagagca gaaggtggca gcatgacacc ggacattatc ctgcagcgta
     1561 ccgggatcga tgtgagagct gtcgaacagg gggatgatgc gtggcacaaa ttacggctcg
     1621 gcgtcatcac cgcttcagaa gttcacaacg tgatagcaaa accccgctcc ggaaagaagt
     1681 ggcctgacat gaaaatgtcc tacttccaca ccctgcttgc tgaggtttgc accggtgtgg
     1741 ctccggaagt taacgctaaa gcactggcct ggggaaaaca gtacgagaac gacgccagaa
     1801 ccctgtttga attcacttcc ggcgtgaatg ttactgaatc cccgatcatc tatcgcgacg
     1861 aaagtatgcg taccgcctgc tctcccgatg gtttatgcag tgacggcaac ggccttgaac
     1921 tgaaatgccc gtttacctcc cgggatttca tgaagttccg gctcggtggt ttcgaggcca
     1981 taaagtcagc ttacatggcc caggtgcagt acagcatgtg ggtgacgcga aaaaatgcct
     2041 ggtactttgc caactatgac ccgcgtatga agcgtgaagg cctgcattat gtcgtgattg
     2101 agcgggatga aaagtacatg gcgagttttg acgagatcgt gccggagttc atcgaaaaaa
     2161 tggacgaggc actggctgaa attggttttg tatttgggga gcaatggcga tgacgcatcc
     2221 tcacgataat atccgggtag gcgcaatcac tttcgtctac tccgttacaa agcgaggctg
     2281 ggtatttccc ggcctttctg ttatccgaaa tccactgaaa gcacagcggc tggctgagga
     2341 gataaataat aaacgagggg ctgtatgcac aaagcatctt ctgttgagtt aagaacgagt
     2401 atcgagatgg cacatagcct tgctcaaatt ggaatcaggt ttgtgccaat accagtagaa
     2461 acagacgaag aatccatggg tatggacagt tttccctttg atatgtaacg gtgaacagtt
     2521 gttctacttt tgtttgttag tcttgatgct tcactgatag atacaagagc cataagaacc
     2581 tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg gttcgttgtt tttgcgtgag
     2641 ccatgagaac gaaccattga gatcatactt actttgcatg tcactcaaaa attttgcctc
     2701 aaaactggtg agctgaattt ttgcagttaa agcatcgtgt agtgtttttc ttagtccgtt
     2761 atgtaggtag gaatctgatg taatggttgt tggtattttg tcaccattca tttttatctg
     2821 gttgttctca agttcggtta cgagatccat ttgtctatct agttcaactt ggaaaatcaa
     2881 cgtatcagtc gggcggcctc gcttatcaac caccaatttc atattgctgt aagtgtttaa
     2941 atctttactt attggtttca aaacccattg gttaagcctt ttaaactcat ggtagttatt
     3001 ttcaagcatt aacatgaact taaattcatc aaggctaatc tctatatttg ccttgtgagt
     3061 tttcttttgt gttagttctt ttaataacca ctcataaatc ctcatagagt atttgttttc
     3121 aaaagactta acatgttcca gattatattt tatgaatttt tttaactgga aaagataagg
     3181 caatatctct tcactaaaaa ctaattctaa tttttcgctt gagaacttgg catagtttgt
     3241 ccactggaaa atctcaaagc ctttaaccaa aggattcctg atttccacag ttctcgtcat
     3301 cagctctctg gttgctttag ctaatacacc ataagcattt tccctactga tgttcatcat
     3361 ctgaacgtat tggttataag tgaacgatac cgtccgttct ttccttgtag ggttttcaat
     3421 cgtggggttg agtagtgcca cacagcataa aattagcttg gtttcatgct ccgttaagtc
     3481 atagcgacta atcgctagtt catttgcttt gaaaacaact aattcagaca tacatctcaa
     3541 ttggtctagg tgattttaat cactatacca attgagatgg gctagtcaat gataattact
     3601 agtccttttc ctttgagttg tgggtatctg taaattctgc tagacctttg ctggaaaact
     3661 tgtaaattct gctagaccct ctgtaaattc cgctagacct ttgtgtgttt tttttgttta
     3721 tattcaagtg gttataattt atagaataaa gaaagaataa aaaaagataa aaagaataga
     3781 tcccagccct gtgtataact cactacttta gtcagttccg cagtattaca aaaggatgtc
     3841 gcaaacgctg tttgctcctc tacaaaacag accttaaaac cctaaaggct taagtagcac
     3901 cctcgcaagc tcggttgcgg ccgcaatcgg gcaaatcgct gaatattcct tttgtctccg
     3961 accatcaggc acctgagtcg ctgtcttttt cgtgacattc agttcgctgc gctcacggct
     4021 ctggcagtga atgggggtaa atggcactac aggcgccttt tatggattca tgcaaggaaa
     4081 ctacccataa tacaagaaaa gcccgtcacg ggcttctcag ggcgttttat ggcgggtctg
     4141 ctatgtggtg ctatctgact ttttgctgtt cagcagttcc tgccctctga ttttccagtc
     4201 tgaccacttc ggattatccc gtgacaggtc attcagactg gctaatgcac ccagtaaggc
     4261 agcggtatca tcaacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4321 ggtcatgaga ttatcagctt tcgctaagga tgatttctgg aattcttccc tatcagtgat
     4381 agagattgac atccctatca gtgatagaga tactgagcac tcctatactt tctagagaat
     4441 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     4501 ggcaccgagt cggtgctttt tttgaagctt gggcccgaac aaaaactcat ctcagaagag
     4561 gatctgaata gcgccgtcga ccatcatcat catcatcatt gagtttaaac ggtctccagc
     4621 ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca
     4681 gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc
     4741 ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg
     4801 cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     4861 tttcgtttta tctgttgttt gtcggtgaac tggatcctta ctcgagtcta gactgcaggc
     4921 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4981 atgagtaaac ttggtctgct gacaggacat tatttgccga ctaccttggt gatctcgcct
     5041 ttcacgtagt ggacaaattc ttccaactga tctgcgcgcg aggccaagcg atcttcttct
     5101 tgtccaagat aagcctgtct agcttcaagt atgacgggct gatactgggc cggcaggcgc
     5161 tccattgccc agtcggcagc gacatccttc ggcgcgattt tgccggttac tgcgctgtac
     5221 caaatgcggg acaacgtaag cactacattt cgctcatcgc cagcccagtc gggcggcgag
     5281 ttccatagcg ttaaggtttc atttagcgcc tcaaatagat cctgttcagg aaccggatca
     5341 aagagttcct ccgccgctgg acctaccaag gcaacgctat gttctcttgc ttttgtcagc
     5401 aagatagcca gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag aatgtcattg
     5461 cgctgccatt ctccaaattg cagttcgcgc ttagctggat aacgccacgg aatgatgtcg
     5521 tcgtgcacaa caatggtgac ttctacagcg cggagaatct cgctctctcc aggggaagcc
     5581 gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag ccttacggtc
     5641 accgtaacca gcaaatcaat atcactgtgt ggcttcaggc cgccatccac tgcggagccg
     5701 tacaaatgta cggccagcaa cgtcggttcg agatggcgct cgatgacgcc aactacctct
     5761 gatagttgag tcgatacttc ggcgatcacc gcttccctca tactcttcct ttttcaatat
     5821 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5881 aaaaataaac aaatagctag ctcactcggt cgctaccagg gttattgtct catgagcgga
     5941 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6001 aaagtgccac ctgcatcgat ttattatgac aacttgacgg ctacatcatt cactttttct
     6061 tcacaaccgg cacggaactc gctcgggctg gccccggtgc attttttaaa tacccgcgag
     6121 aagtagagtt gatcgtcaaa accaacattg cgaccgacgg tggcgatagg catccgggtg
     6181 gtgctcaaaa gcagcttcgc ctggctgata cgttggtcct cgcgccagct taagacgcta
     6241 atccctaact gctggcggaa aagatgtgac agacgcgacg gcgacaagca aacatgctgt
     6301 gcgacgctgg cgatatcaaa attgctgtct gccaggtgat cgctgatgta ctgacaagcc
     6361 tcgcgtaccc gattatccat cggtggatgg agcgactcgt taatcgcttc catgcgccgc
     6421 agtaacaatt gctcaagcag atttatcgcc agcagctccg aatagcgccc ttccccttgc
     6481 ccggcgttaa tgatttgccc aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg
     6541 cgaaagaacc ccgtattggc aaatattgac ggccagttaa gccattcatg ccagtaggcg
     6601 cgcggacgaa agtaaaccca ctggtgatac cattcgcgag cctccggatg acgaccgtag
     6661 tgatgaatct ctcctggcgg gaacagcaaa atatcacccg gtcggcaaac aaattctcgt
     6721 ccctgatttt tcaccacccc ctgaccgcga atggtgagat tgagaatata acctttcatt
     6781 cccagcggtc ggtcgataaa aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc
     6841 gccaccagat gggcattaaa cgagtatccc ggcagcaggg gatcattttg cgcttcagcc
     6901 atacttttca tactcccgcc attcagag
//
