LOCUS       62056_10955        7224 bp DNA     circular SYN 02-NOV-2020
DEFINITION  Cloning vector pFR56, complete sequence.
ACCESSION   MT412099
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7224)
  AUTHORS   Rousset F, Fernandez-Rodriguez J, Rocha E, Cabezas-Caballero J, 
            Piastra-Facon F, Clermont O, Denamur E, Bikard D.
  TITLE     Parallel CRISPRi screening of the E. coli core genome reveals a 
            modulation of gene essentiality by horizontal gene transfer
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7224)
  AUTHORS   Rousset F, Fernandez-Rodriguez J, Piastra-Facon F, Bikard D.
  TITLE     Direct Submission
  JOURNAL   Submitted (29-APR-2020) Synthetic Biology Group, Institut Pasteur, 
            28 rue du Dr Roux, Paris 75015, Paris
REFERENCE   3  (bases 1 to 7224)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (29-APR-2020) Synthetic Biology Group, Institut Pasteur, 28 rue du 
            Dr Roux, Paris 75015, Paris"
FEATURES             Location/Qualifiers
     source          1..7224
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      38..73
                     /label=J23110
                     /note="J23110"
                     /regulatory_class="promoter"
     regulatory      78..93
                     /label=B0030 RBS
                     /note="B0030 RBS"
                     /regulatory_class="ribosome_binding_site"
     CDS             99..701
                     /codon_start=1
                     /transl_table=11
                     /product="PhlF repressor"
                     /label=PhlF repressor
                     /protein_id="QOT13791.1"
                     /translation="MARTPSRSSIGSLRSPHTHKAILTSTIEILKECGYSGLSIESVAR
                     RAGASKPTIYRWWTNKAALIAEVYENESEQVRKFPDLGSFKADLDFLLRNLWKVWRETI
                     CGEAFRCVIAEAQLDPATLTQLKDQFMERRREMPKKLVENAISNGELPKDTNRELLLDM
                     IFGFCWYRLLTEQLTVEQDIEEFTFLLINGVCPGTQR"
     regulatory      712..769
                     /label=modified L3S2P11 terminator
                     /note="modified L3S2P11 terminator"
                     /regulatory_class="terminator"
     misc_feature    781..832
                     /label=PhlF promoter
                     /note="PhlF promoter"
     regulatory      797..802
                     /regulatory_class="minus_35_signal"
     misc_feature    802..832
                     /label=phO
                     /note="phO"
     regulatory      820..826
                     /regulatory_class="minus_10_signal"
     misc_feature    832..867
                     /label=RBS
                     /note="RBS"
     CDS             867..4970
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     regulatory      4984..5038
                     /label=terminator ECK120010818
                     /note="terminator ECK120010818"
                     /regulatory_class="terminator"
     misc_feature    5053..5277
                     /label=lambda cos
                     /note="lambda cos"
     misc_feature    complement(5178..5210)
                     /label=cosN site
                     /note="cosN site"
     misc_RNA        complement(5305..5380)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    complement(5381..5401)
                     /label=control spacer with 2 BsaI
                     /note="control spacer with 2 BsaI"
     promoter        complement(5401..5435)
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological
                     Parts BBa_J23119) modified to end with an SpeI site"
     regulatory      complement(5470..5527)
                     /label=terminator ECK120033737
                     /note="terminator ECK120033737"
                     /regulatory_class="terminator"
     CDS             complement(5538..6194)
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     regulatory      complement(6195..6220)
                     /label=synthetic RBS
                     /note="synthetic RBS"
                     /regulatory_class="ribosome_binding_site"
     oriT            complement(6334..6443)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     regulatory      complement(6597..6610)
                     /label=T7 promoter
                     /note="T7 promoter"
                     /regulatory_class="promoter"
     rep_origin      complement(6643..7188)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
ORIGIN
        1 atttcagtgc aatttatctc ttcaaatgta gcacgatttt acggctagct cagtcctagg
       61 tacaatgcta gcgaatcatt aaagaggaga aaggtactat ggcacgtacc ccgtcacgta
      121 gtagcattgg tagcctgcgt agtccgcata cccataaagc aattctgacc agtaccatcg
      181 agatcctgaa agaatgtggt tatagcggac tgagcattga aagcgttgca cgtcgtgccg
      241 gagcaagcaa accgaccatt tatcgttggt ggacgaataa agcagcactg attgccgaag
      301 tgtatgaaaa tgaaagcgaa caggtgcgta aatttccgga tctgggtagc tttaaagcag
      361 atctggattt tttactgcgt aatttatgga aagtttggcg tgaaactatt tgcggtgaag
      421 catttcgttg tgttattgca gaagctcagc tggatcctgc aaccctgacc cagttaaagg
      481 atcaatttat ggaacgtcgt cgtgagatgc cgaaaaaact ggttgaaaat gccattagca
      541 atggtgaact gccgaaagat accaatcgtg aacttcttct ggatatgatt tttggttttt
      601 gttggtatcg cctgttaacc gaacagctga ccgttgaaca ggatattgaa gaatttacct
      661 tccttctgat taatggtgtt tgtccgggta ctcagcgtta actaggccat aatcgctacc
      721 aaattccaga aaacagacgc tttcgagcgt cttttttcgt tttggtcacg acgtactgaa
      781 tctgattcgt taccaattga catgatacga aacgtaccgt atcgttaagg tggaggcata
      841 tcaaaggacg agtgcaggtg gcaaaaatgg ataagaaata cagcataggc ttagctatcg
      901 gcacaaatag cgtcggatgg gcggtgatca ctgatgaata taaagttccg tctaaaaagt
      961 tcaaggtact gggtaataca gatcgccata gtatcaaaaa gaacttaatc ggtgcgcttc
     1021 tgttcgattc cggcgaaacc gcagaagcaa cacgtctgaa acgcaccgct cgtcgccgtt
     1081 acacccgtcg taaaaaccgc atctgctacc tgcaagaaat cttctctaac gaaatggcta
     1141 aagtagatga cagctttttt caccgtctgg aagaatcatt tctggtggaa gaagataaaa
     1201 agcacgaacg tcatccaatc ttcggcaaca ttgtggacga agtagcgtat cacgaaaaat
     1261 acccgactat ctatcacctg cgcaaaaagc tggtcgattc gacggataag gccgatctgc
     1321 gtctgatcta tctggcctta gcgcatatga ttaagttccg tggtcatttc ctgatcgaag
     1381 gcgacctgaa tccagacaac agcgatgtag acaaactgtt catccagctg gtgcaaacct
     1441 ataaccagct gtttgaagaa aacccaatta atgctagcgg tgttgacgcg aaagcgatct
     1501 tgtccgcacg cctgtccaaa tcccgtcgtc tggaaaactt aattgcgcaa ctgccgggtg
     1561 agaagaaaaa cggactgttc ggcaatctga tcgctcttag cttgggactg accccgaact
     1621 tcaaaagcaa cttcgatctg gcagaggacg caaaacttca acttagcaaa gatacgtatg
     1681 acgatgactt ggataactta ctggcccaga tcggagatca gtacgctgat ctgtttctgg
     1741 cggcaaagaa cttatcagac gctattctcc tgtctgatat tcttcgtgtg aataccgaaa
     1801 tcaccaaagc accgctttct gcatccatga ttaaacgcta tgacgaacat caccaagatc
     1861 tgactcttct gaaagcgctg gtacggcaac aactgccgga gaagtacaag gagatcttct
     1921 ttgaccaatc caaaaacggc tacgcgggtt atattgacgg gggtgcaagc caagaggagt
     1981 tctacaaatt catcaagcca atcttagaaa aaatggatgg cacggaagaa ttacttgtta
     2041 aactgaatcg tgaggatctg cttcgtaaac agcgtacctt cgacaacggt agcattccgc
     2101 accagatcca cttaggtgaa ctgcacgcta tcctgcgtcg ccaagaggat ttttacccgt
     2161 tcctgaaaga taatcgtgaa aaaatcgaaa aaatcctgac ctttcgtatc ccgtattatg
     2221 tcggcccgct ggcgcgtggc aactcccgtt tcgcgtggat gactcgcaaa tccgaagaaa
     2281 ctattacccc gtggaacttc gaggaagtgg ttgacaaagg cgcaagcgcc caatccttca
     2341 tcgagcgcat gactaacttt gataaaaacc tgccgaacga aaaggtactg ccgaaacact
     2401 cccttctgta cgaatacttc accgtgtaca acgagctgac taaagtaaag tatgtgactg
     2461 agggcatgcg taaacctgca ttcctgagcg gtgaacagaa aaaagcaatt gttgatttac
     2521 tgtttaaaac caaccgtaaa gtaaccgtta aacagctgaa agaggactac ttcaagaaaa
     2581 tcgaatgctt cgactccgtc gagattagtg gagttgaaga tcgttttaat gcaagtttag
     2641 gcacgtatca cgatttatta aagatcatta aagacaaaga tttcttggac aacgaagaaa
     2701 atgaggacat cttagaggac atcgtcctga ccctgactct gttcgaagat cgtgaaatga
     2761 ttgaagaacg cttgaagacg tatgctcacc tgtttgacga taaagtaatg aaacaactga
     2821 aacgtcgccg ttatactggc tggggccgtc tgagccgtaa actgattaac ggtatccgtg
     2881 acaaacagtc cggtaaaact attctggact tcctgaaatc tgacggcttc gcaaaccgta
     2941 acttcatgca actgattcac gacgattccc tgaccttcaa agaggacatc cagaaagctc
     3001 aggtttctgg tcaaggtgat tctctgcacg agcatatcgc caatttagca ggtagtccgg
     3061 cgatcaaaaa aggtatcctg caaaccgtga aagtggtgga tgagcttgtg aaagttatgg
     3121 gtcgtcacaa accggaaaac attgttatcg agatggctcg tgaaaaccaa acgacccaga
     3181 agggacagaa aaactcccgc gaacgcatga aacgtatcga ggagggtatt aaagaacttg
     3241 gctctcagat tctgaaagaa caccctgttg aaaataccca actgcaaaat gaaaaactgt
     3301 acctgtacta cctgcaaaat ggtcgtgaca tgtatgtaga tcaggagctg gacatcaacc
     3361 gcctctccga ttacgacgtt gacgccattg ttccgcagtc ttttctgaaa gatgattcca
     3421 ttgataacaa agtactcacc cgtagcgata aaaaccgtgg gaagagtgac aacgttccat
     3481 cggaagaagt agttaagaaa atgaagaact attggcgtca actgcttaac gcgaaactga
     3541 ttactcaacg taaatttgat aacctgacca aagctgaacg tggcggtttg tctgagctgg
     3601 ataaggcggg ttttattaaa cgtcaactgg tagaaactcg ccagattaca aaacatgttg
     3661 ctcagattct ggactctcgt atgaacacta aatacgatga aaatgacaaa ctgatccgcg
     3721 aagttaaggt tattaccctg aaatctaagc tggtttccga cttccgtaaa gatttccaat
     3781 tctataaagt gcgcgagatt aacaactatc accacgcgca cgacgcatat ctgaatgcag
     3841 ttgttggcac ggcactgatc aaaaaatatc cgaaactgga aagcgaattt gtgtacggcg
     3901 attataaagt ttacgacgtg cgcaaaatga tcgccaaatc tgaacaggaa attggcaaag
     3961 caaccgctaa atactttttc tactcaaaca ttatgaattt cttcaaaacc gaaatcacct
     4021 tagcgaatgg cgaaattcgt aaacgccctc tgatcgaaac caacggcgaa acgggtgaga
     4081 tcgtgtggga caaaggtcgt gatttcgcta ctgtccgcaa agttctgtcc atgcctcaag
     4141 taaacatcgt taaaaagact gaggtacaga ctggcggttt cagcaaggaa tccattctgc
     4201 cgaaacgcaa ctccgacaaa ctgatcgcgc gtaagaaaga ctgggatccg aagaaatacg
     4261 gtggcttcga ttctccaacc gtggcataca gcgttctggt agtcgccaaa gtcgaaaagg
     4321 gtaaatcaaa aaaactgaaa tcagtgaaag aacttttagg catcaccatt atggaacgta
     4381 gctctttcga aaaaaacccg attgacttcc tcgaagcgaa ggggtacaag gaagtaaaga
     4441 aagatctgat tatcaaactg ccgaagtatt ccctgttcga actggaaaat ggtcgtaaac
     4501 gtatgttagc gtctgcgggt gaactgcaaa aagggaacga attggccctt ccgtccaagt
     4561 acgtgaactt cctgtatctg gcctcgcact acgagaaact gaaaggtagt ccggaagata
     4621 atgagcagaa acagctgttc gtggaacagc acaaacacta tctggacgag attattgaac
     4681 agatttctga gtttagcaaa cgcgtaattc tggcggacgc gaatctggat aaagtcctga
     4741 gcgcctacaa taaacaccgt gataaaccga tccgtgaaca ggcagaaaac atcattcacc
     4801 tgttcacgct gactaatctt ggtgctccgg cagccttcaa atacttcgac accacgatcg
     4861 atcgtaaacg ttacacctcc actaaagaag tcttagatgc aactcttatt caccagagca
     4921 tcactggcct gtatgaaact cgtattgatc tgagtcagtt gggcggtgac taataacgtt
     4981 aaagtcagtt tcacctgttt tacgttaaaa cccgcttcgg cgggttttta cttttgggtt
     5041 tagccgaacg ccccaaaaag cctcgctttc agcacctgtc gtttcctttc ttttcagagg
     5101 gtattttaaa taaaaacatt aagttatgac gaagaagaac ggaaacgcct taaaccggaa
     5161 aattttcata aatagcgaaa acccgcgagg tcgccgcccc gtaacctgtc ggatcaccgg
     5221 aaaggacccg taaagtgata atgattatca tctacatatc acaacgtgcg taaagggact
     5281 agtggatgtt tgatctcaaa aaaagcaccg actcggtgcc actttttcaa gttgataacg
     5341 gactagcctt attttaactt gctatttcta gctctaaaac cgagacctag actggtctca
     5401 actagtatta tacctaggac tgagctagct gtcagtcggg tagcaccaga agtctatagc
     5461 atgtgcatac ctttggtcga aaaaaaaagc ccgcactgtc aggtgcgggc ttttttcagt
     5521 gtttccttgc cggattacgc cccgccctgc cactcatcgc agtattgttg taattcatta
     5581 agcattctgc cgacatggaa gccatcacaa acggcatgat gaacttggat cgccagtggc
     5641 attaacacct tgtcgccttg cgtataatat tttcccatag tgaaaacggg ggcgaagaag
     5701 ttgtccatat ttgctacgtt taaatcaaaa ctggtgaaac tcacccacgg attggcactg
     5761 acgaaaaaca tattttcgat aaacccttta gggaaatatg ctaagttttc accgtaacac
     5821 gccacatctt gactatatat gtgtagaaac tgccggaaat cgtcgtggta ttctgaccag
     5881 agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc
     5941 catatcacca gctcaccgtc tttcattgcc atacgaaact ccggatgtgc attcatcagg
     6001 cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggttttt
     6061 aaaaaggccg taatatccag ctgaacggtt tggttatagg tgcactgagc aactgactgg
     6121 aatgcctcaa aatgttcttt acgatgccat tgacttatat caactgtagt atatccagtg
     6181 atttttttct ccattttagc ttccttagct tgcgaaatct cgataactca aaaaatagta
     6241 gtgatcttat ttcattatgg tgaaagttgt cttacgtgca acattttcgc aaaaagttgg
     6301 cgctttatca acactgtccc tcctgttcag ctaccggcca gcctcgcaga gcaggattcc
     6361 cgttgagcac cgccaggtgc gaataaggga cagtgaagaa ggaacacccg ctcgcgggtg
     6421 ggcctacttc acctatcctg cccggctgac gccgttggat acaccaagga aagtctacac
     6481 gaaccctttg gcaaaatcct gtatatcgtg cgaaaaagga tggatatacc gaaaaaatcg
     6541 ctataatgac cccgaagcag ggttatgcag cggaaaagcg ctggtaccca attcgcccta
     6601 tagtgagtct cctggaagtg agagggccgc ggcaaagccg tttttccata ggctccgccc
     6661 ccctgacaag catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc tgacaggact
     6721 ataaagatac caggcgtttc cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt
     6781 tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg tctcattcca cgcctgacac
     6841 tcagttccgg gtaggcagtt cgctccaagc tggactgtat gcacgaaccc cccgttcagt
     6901 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggaa agacatgcaa
     6961 aagcaccact ggcagcagcc actggtaatt gatttagagg agttagtctt gaagtcatgc
     7021 gccggttaag gctaaactga aaggacaagt tttggtgact gcgctcctcc aagccagtta
     7081 cctcggttca aagagttggt agctcagaga accttcgaaa aaccgccctg caaggcggtt
     7141 ttttcgtttt cagagcaaga gattacgcgc agaccaaaac gatctcaaga agatcatctt
     7201 attaatcaga taaaatattt ctag
//
