LOCUS       62056_4905        9764 bp DNA     circular SYN 26-MAY-2020
DEFINITION  Cloning vector pCasCure-Rif, complete sequence.
ACCESSION   MT262893
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9764)
  AUTHORS   Chen L.
  TITLE     CRISPR/Cas9-mediated plasmid curation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9764)
  AUTHORS   Chen L.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-MAR-2020) Center for Discovery and Innovation, 
            Hackensack-Meridian Health, 340 Kingsland Street, Nutley, NJ 07110, 
            USA
REFERENCE   3  (bases 1 to 9764)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (27-MAR-2020) Center for Discovery and Innovation, 
            Hackensack-Meridian Health, 340 Kingsland Street, Nutley, NJ 07110, 
            USA"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..9764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /label=AmpR promoter
     primer_bind     579..595
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     633..649
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(714..2132)
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     promoter        2518..2552
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        2570..2645
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2678..2694)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2707..6810)
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     promoter        complement(6838..7122)
                     /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)"
     CDS             7149..8024
                     /label=araC
                     /note="L-arabinose regulatory protein"
     primer_bind     complement(8151..8167)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(8175..8191)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(8199..8229)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(8244..8265)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(8553..9141)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(9312..9764)
                     /codon_start=1
                     /transl_table=11
                     /product="Arr"
                     /label=Arr
                     /protein_id="QJX58432.1"
                     /translation="MVKDWIPISHDNYKQVQGPFYHGTKANLAIGDLLTTGFISHFEDG
                     RILKHIYFSALMEPAVWGAELAMSLSGLEGRGYIYIVEPTGPFEDDPNLTNKRFPGNPT
                     QSYRTCEPLRIVGVVEDWEGHPVELIRGMLDSLEDLKRRGLHVIED"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
      181 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
      241 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
      301 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
      361 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
      421 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
      481 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
      541 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
      601 gacgcgtatt gggatggtac cgggcccccc ctcgaggtcg acggtatcga tacggcatca
      661 gagcagattg tactgagagt gcaccataat cggcattttc ttttgcgttt ttatttgtta
      721 actgttaatt gtccttgttc aaggatgctg tctttgacaa cagatgtttt cttgcctttg
      781 atgttcagca ggaagctagg cgcaaacgtt gattgtttgt ctgcgtagaa tcctctgttt
      841 gtcatatagc ttgtaatcac gacattgttt cctttcgctt gaggtacagc gaagtgtgag
      901 taagtaaagg ttacatcgtt aggatcaaga tccattttta acacaaggcc agttttgttc
      961 agcggcttgt atgggccagt taaagaatta gaaacataac caagcatgta aatatcgtta
     1021 gacgtaatgc cgtcaatcgt catttttgat ccgcgggagt cagtgaacag ataccatttg
     1081 ccgttcattt taaagacgtt cgcgcgttca atttcatctg ttactgtgtt agatgcaatc
     1141 agcggtttca tcactttttt cagtgtgtaa tcatcgttta gctcaatcat accgagagcg
     1201 ccgtttgcta actcagccgt gcgtttttta tcgctttgca gaagtttttg actttcttga
     1261 cggaagaatg atgtgctttt gccatagtat gctttgttaa ataaagattc ttcgccttgg
     1321 tagccatctt cagttccagt gtttgcttca aatactaagt atttgtggcc tttatcttct
     1381 acgtagtgag gatctctcag cgtatggttg tcgcctgagc tgtagttgcc ttcatcgatg
     1441 aactgctgta cattttgata cgtttttccg tcaccgtcaa agattgattt ataatcctct
     1501 acaccgttga tgttcaaaga gctgtctgat gctgatacgt taacttgtgc agttgtcagt
     1561 gtttgtttgc cgtaatgttt accggagaaa tcagtgtaga ataaacggat ttttccgtca
     1621 gatgtaaatg tggctgaacc tgaccattct tgtgtttggt cttttaggat agaatcattt
     1681 gcatcgaatt tgtcgctgtc tttaaagacg cggccagcgt ttttccagct gtcaatagaa
     1741 gtttcgccga ctttttgata gaacatgtaa atcgatgtgt catccgcatt tttaggatct
     1801 ccggctaatg caaagacgat gtggtagccg tgatagtttg cgacagtgcc gtcagcgttt
     1861 tgtaatggcc agctgtccca aacgtccagg ccttttgcag aagagatatt tttaattgtg
     1921 gacgaatcga actcaggaac ttgatatttt tcattttttt gctgttcagg gatttgcagc
     1981 atatcatggc gtgtaatatg ggaaatgccg tatgtttcct tatatggctt ttggttcgtt
     2041 tctttcgcaa acgcttgagt tgcgcctcct gccagcagtg cggtagtaaa ggttaatact
     2101 gttgcttgtt ttgcaaactt tttgatgttc atcgttcatg tctccttttt tatgtactgt
     2161 gttagcggtc tgcttcttcc agccctcctg tttgaagatg gcaagttagt tacgcacaat
     2221 aaaaaaagac ctaaaatatg taaggggtga cgccaaagta tacactttgc cctttacaca
     2281 ttttaggtct tgcctgcttt atcagtaaca aacccgcgcg atttactttt cgacctcatt
     2341 ctattagact ctcgtttgga ttgcaactgg tctattttcc tcttttgttt gatagaaaat
     2401 cataaaagga tttgcagact acgggcctaa agaactaaaa aatctatctg tttcttttca
     2461 ttctctgtat tttttatagt ttctgttgca tgggcataaa gttgcaagct tgatatcttg
     2521 acagctagct cagtcctagg tataatacta gtcgagacca ttggtctcag ttttagagct
     2581 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     2641 ggtgcttttt ttgatatcga attcctgcag cccgggggat ccactagttc tagaaaccag
     2701 ccatcagtca cctcctagct gactcaaatc aatgcgtgtt tcataaagac cagtgatgga
     2761 ttgatggata agagtggcat ctaaaacttc ttttgtagac gtatatcgtt tacgatcaat
     2821 tgttgtatca aaatatttaa aagcagcggg agctccaaga ttcgtcaacg taaataaatg
     2881 aataatattt tctgcttgtt cacgtattgg tttgtctcta tgtttgttat atgcactaag
     2941 aactttatct aaattggcat ctgctaaaat aacacgctta gaaaattcac tgatttgctc
     3001 aataatctca tctaaataat gcttatgctg ctccacaaac aattgttttt gttcgttatc
     3061 ttctggacta cccttcaact tttcataatg actagctaaa tataaaaaat tcacatattt
     3121 gcttggcaga gccagctcat ttcctttttg taattctccg gcactagcca gcatccgttt
     3181 acgaccgttt tctaactcaa aaagactata tttaggtagt ttaatgatta agtctttttt
     3241 aacttcctta tatcctttag cttctaaaaa gtcaatcgga tttttttcaa aggaacttct
     3301 ttccataatt gtgatcccta gtaactcttt aacggatttt aacttcttcg atttcccttt
     3361 ttccacctta gcaaccacta ggactgaata agctaccgtt ggactatcaa aaccaccata
     3421 tttttttgga tcccagtctt ttttacgagc aataagcttg tccgaatttc tttttggtaa
     3481 aattgactcc ttggagaatc cgcctgtctg tacttctgtt ttcttgacaa tattgacttg
     3541 gggcatggac aatactttgc gcactgtggc aaaatctcgc cctttatccc agacaatttc
     3601 tccagtttcc ccattagttt cgattagagg gcgtttgcga atctctccat ttgcaagtgt
     3661 aatttctgtt ttgaagaagt tcatgatatt agagtaaaag aaatattttg cggttgcttt
     3721 gcctatttct tgctcagact tagcaatcat tttacgaaca tcataaactt tataatcacc
     3781 atagacaaac tccgattcaa gttttggata tttcttaatc aaagcagttc caacgacggc
     3841 atttagatac gcatcatggg catgatggta attgttaatc tcacgtactt tatagaattg
     3901 gaaatctttt cggaagtcag aaactaattt agattttaag gtaatcactt taacctctcg
     3961 aataagttta tcattttcat cgtatttagt attcatgcga ctatccaaaa tttgtgccac
     4021 atgcttagtg atttggcgag tttcaaccaa ttggcgtttg ataaaaccag ctttatcaag
     4081 ttcactcaaa cctccacgtt cagctttcgt taaattatca aacttacgtt gagtgattaa
     4141 cttggcgttt agaagttgtc tccaatagtt tttcatcttt ttgactactt cttcacttgg
     4201 aacgttatcc gatttaccac gatttttatc agaacgcgtt aagaccttat tgtctattga
     4261 atcgtcttta aggaaacttt gtggaacaat gtgatcgaca tcataatcac ttaaacgatt
     4321 aatatctaat tcttggtcca catacatgtc tcttccattt tggagataat agagatagag
     4381 cttttcattt tgcaattgag tattttcaac aggatgctct ttaagaatct gacttcctaa
     4441 ttctttgata ccttcttcga ttcgtttcat acgctctcgc gaatttttct ggcccttttg
     4501 agttgtctga ttttcacgtg ccatttcaat aacgatattt tctggcttat gccgccccat
     4561 tactttgacc aattcatcaa caacttttac agtctgtaaa ataccttttt taatagcagg
     4621 gctaccagct aaatttgcaa tatgttcatg taaactatcg ccttgtccag acacttgtgc
     4681 tttttgaatg tcttctttaa atgtcaaact atcatcatgg atcagctgca taaaattgcg
     4741 attggcaaaa ccatctgatt tcaaaaaatc taatattgtt ttgccagatt gcttatccct
     4801 aataccatta atcaattttc gagacaaacg tccccaacca gtataacggc gacgtttaag
     4861 ctgtttcatc accttatcat caaagaggtg agcatatgtt ttaagtcttt cctcaatcat
     4921 ctccctatct tcaaataagg tcaatgttaa aacaatatcc tctaagatat cttcattttc
     4981 ttcattatcc aaaaaatctt tatctttaat aatttttagc aaatcatggt aggtacctaa
     5041 tgaagcatta aatctatctt caactcctga aatttcaaca ctatcaaaac attctatttt
     5101 tttgaaataa tcttctttta attgcttaac ggttactttt cgatttgttt tgaagagtaa
     5161 atcaacaatg gctttcttct gttcacctga aagaaatgct ggttttcgca ttccttcagt
     5221 aacatatttg acctttgtca attcgttata aaccgtaaaa tactcataaa gcaaactatg
     5281 ttttggtagt actttttcat ttggaagatt tttatcaaag tttgtcatgc gttcaataaa
     5341 tgattgagct gaagcacctt tatcgacaac ttcttcaaaa ttccatgggg taattgtttc
     5401 ttcagacttc cgagtcatcc atgcaaaacg actattgcca cgcgccaatg gaccaacata
     5461 ataaggaatt cgaaaagtca agattttttc aatcttctca cgattgtctt ttaaaaatgg
     5521 ataaaagtct tcttgtcttc tcaaaatagc atgcagctca cccaagtgaa tttgatgggg
     5581 aatagagccg ttgtcaaagg tccgttgctt gcgcagcaaa tcttcacgat ttagtttcac
     5641 caataattcc tcagtaccat ccattttttc taaaattggt ttgataaatt tataaaattc
     5701 ttcttggcta gctcccccat caatataacc tgcatatccg ttttttgatt gatcaaaaaa
     5761 gatttcttta tacttttctg gaagttgttg tcgaactaaa gcttttaaaa gagtcaagtc
     5821 ttgatgatgt tcatcgtagc gtttaatcat tgaagctgat aggggagcct tagttatttc
     5881 agtatttact cttaggatat ctgaaagtaa aatagcatct gataaattct tagctgccaa
     5941 aaacaaatca gcatattgat ctccaatttg cgccaataaa ttatctaaat catcatcgta
     6001 agtatctttt gaaagctgta atttagcatc ttctgccaaa tcaaaatttg atttaaaatt
     6061 aggggtcaaa cccaatgaca aagcaatgag attcccaaat aagccatttt tcttctcacc
     6121 ggggagctga gcaatgagat tttctaatcg tcttgattta ctcaatcgtg cagaaagaat
     6181 cgctttagca tctactccac ttgcgttaat agggttttct tcaaataatt gattgtaggt
     6241 ttgtaccaac tggataaata gtttgtccac atcactatta tcaggattta aatctccctc
     6301 aatcaaaaaa tgaccacgaa acttaatcat atgcgctaag gccaaataga ttaagcgcaa
     6361 atccgcttta tcagtagaat ctaccaattt ttttcgcaga tgatagatag ttggatattt
     6421 ctcatgataa gcaacttcat ctactatatt tccaaaaata ggatgacgtt catgcttctt
     6481 gtcttcttcc accaaaaaag actcttcaag tcgatgaaag aaactatcat ctactttcgc
     6541 catctcattt gaaaaaatct cctgtagata acaaatacga ttcttccgac gtgtatacct
     6601 tctacgagct gtccgtttga gacgagtcgc ttccgctgtc tctccactgt caaataaaag
     6661 agcccctata agattttttt tgatactgtg gcggtctgta tttcccagaa ccttgaactt
     6721 tttagacgga accttatatt catcagtgat caccgcccat ccgacgctat ttgtgccgat
     6781 atctaagcct attgagtatt tcttatccat taaatagctc ctgggcggcc gccgggtatg
     6841 gagaaacagt agagagttgc gataaaaagc gtcaggtagg atccgctaat cttatggata
     6901 aaaatgctat ggcatagcaa agtgtgacgc cgtgcaaata atcaatgtgg acttttctgc
     6961 cgtgattata gacacttttg ttacgcgttt ttgtcatggc tttggtcccg ctttgttaca
     7021 gaatgctttt aataagcggg gttaccggtt tggttagcga gaagagccag taaaagacgc
     7081 agtgacggca atgtctgatg caatatggac aattggtttc ttctctgaat ggcgggagta
     7141 tgaaaagtat ggctgaagcg caaaatgatc ccctgctgcc gggatactcg tttaatgccc
     7201 atctggtggc gggtttaacg ccgattgagg ccaacggtta tctcgatttt tttatcgacc
     7261 gaccgctggg aatgaaaggt tatattctca atctcaccat tcgcggtcag ggggtggtga
     7321 aaaatcaggg acgagaattt gtttgccgac cgggtgatat tttgctgttc ccgccaggag
     7381 agattcatca ctacggtcgt catccggagg ctcgcgaatg gtatcaccag tgggtttact
     7441 ttcgtccgcg cgcctactgg catgaatggc ttaactggcc gtcaatattt gccaatacgg
     7501 ggttctttcg cccggatgaa gcgcaccagc cgcatttcag cgacctgttt gggcaaatca
     7561 ttaacgccgg gcaaggggaa gggcgctatt cggagctgct ggcgataaat ctgcttgagc
     7621 aattgttact gcggcgcatg gaagcgatta acgagtcgct ccatccaccg atggataatc
     7681 gggtacgcga ggcttgtcag tacatcagcg atcacctggc agacagcaat tttgatatcg
     7741 ccagcgtcgc acagcatgtt tgcttgtcgc cgtcgcgtct gtcacatctt ttccgccagc
     7801 agttagggat tagcgtctta agctggcgcg aggaccaacg tatcagccag gcgaagctgc
     7861 ttttgagcac cacccggatg cctatcgcca ccgtcggtcg caatgttggt tttgacgatc
     7921 aactctattt ctcgcgggta tttaaaaaat gcaccggggc cagcccgagc gagttccgtg
     7981 ccggttgtga agaaaaagtg aatgatgtag ccgtcaagtt gtcataattg gtaacgaatc
     8041 agacaattga cggcttgacg gagtagcata gggtttgcag aatccctgct tcgcggccgc
     8101 caccgcggtg gagctcatcc caatggcgcg ccgagcttgg ctcgagcatg gtcatagctg
     8161 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     8221 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     8281 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     8341 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     8401 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     8461 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     8521 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     8581 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     8641 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     8701 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     8761 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     8821 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     8881 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     8941 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     9001 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     9061 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     9121 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     9181 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     9241 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     9301 tggtctgaca gctagtcttc aatgacgtgt aaaccacggc gctttaagtc ctccaacgaa
     9361 tccaacattc cccttattaa ttcaacagga tgcccctccc agtcttcaac aacgccaaca
     9421 attctcaagg gttcgcaggt tctataggac tgtgttggat taccgggaaa tcttttgttc
     9481 gtaagattcg gatcgtcttc gaacggtcct gttggctcaa ctatgtatat gtagccgcga
     9541 ccctcgaggc cagacagtga catagcaagc tcagctcccc aaactgctgg ctccatcaag
     9601 gctgaaaagt agatgtgctt aagaatacga ccgtcctcga aatgagagat gaaccctgtg
     9661 gttagcaagt caccaatcgc caaattggct ttggttccat gatagaacgg tccttgcacc
     9721 tgcttgtaat tatcatgaga gatgggaatc caatctttta ccat
//
