LOCUS       62056_1980        9989 bp DNA     circular SYN 25-JAN-2019
DEFINITION  Cloning vector p2U6-CAG-Cas9-p2A-puro, complete sequence.
ACCESSION   MK318530
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9989)
  AUTHORS   Kim J.
  TITLE     Direct Submission
  JOURNAL   Submitted (18-DEC-2018) Biological Sciences, UCSD, 9500 Gilman Dr, 
            La Jolla, CA 92093, USA
REFERENCE   2  (bases 1 to 9989)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Submitted 
            (18-DEC-2018) Biological Sciences, UCSD, 9500 Gilman Dr, La Jolla, 
            CA 92093, USA"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..9989
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2..13
                     /label=Factor Xa site
                     /note="Factor Xa recognition and cleavage site"
     regulatory      21..261
                     /label=RNA pol lll promoter for human U6 snRNA
                     /note="RNA pol lll promoter for human U6 snRNA"
                     /regulatory_class="promoter"
     misc_RNA        294..369
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        423..663
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     intron          1442..2454
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and
                     rabbit beta-globin"
     CDS             2647..6750
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             6790..6846
                     /label=P2A
                     /note="2A peptide from porcine teschovirus-1 polyprotein"
     CDS             6847..7440
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
     polyA_signal    7497..7630
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(7715..7745)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(7760..7781)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(8069..8657)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8831..9688)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(9689..9793)
                     /label=AmpR promoter
ORIGIN
        1 aattgaaggt cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac
       61 gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag
      121 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
      181 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
      241 tatatatctt gtggaaagga cgaaacaccg tgcacgatag actaccgtat ctagttttag
      301 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      361 agtcggtgct ttttttgttt aaacacacgg ttcctagtat cggtccgaag gtcgggcagg
      421 aagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
      481 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
      541 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
      601 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
      661 gacgaaacac cgcctacgtc agtacctaac agtgttttag agctagaaat agcaagttaa
      721 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttacgc
      781 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      841 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      901 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      961 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
     1021 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     1081 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     1141 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
     1201 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
     1261 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
     1321 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
     1381 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
     1441 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
     1501 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
     1561 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
     1621 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
     1681 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
     1741 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1801 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1861 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1921 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc
     1981 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     2041 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     2101 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     2161 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     2221 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     2281 cttcgtgcgt cgccgcgccg ccgtcccctt ctccatctcc agcctcgggg ctgtccgcag
     2341 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     2401 ggcggctcta gtgcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     2461 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattctc tagagatatc
     2521 gctagcacca tggactacaa agaccatgac ggtgattata aagatcatga catcgattac
     2581 aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggcat tcaccgcggg
     2641 gtacccatgg acaagaagta ctccattggg ctcgatatcg gcacaaacag cgtcggctgg
     2701 gccgtcatta cggacgagta caaggtgccg agcaaaaaat tcaaagttct gggcaatacc
     2761 gatcgccaca gcataaagaa gaacctcatt ggcgccctcc tgttcgactc cggggagacg
     2821 gccgaagcca cgcggctcaa aagaacagca cggcgcagat atacccgcag aaagaatcgg
     2881 atctgctacc tgcaggagat ctttagtaat gagatggcta aggtggatga ctctttcttc
     2941 cataggctgg aggagtcctt tttggtggag gaggataaaa agcacgagcg ccacccaatc
     3001 tttggcaata tcgtggacga ggtggcgtac catgaaaagt acccaaccat atatcatctg
     3061 aggaagaagc ttgtagacag tactgataag gctgacttgc ggttgatcta tctcgcgctg
     3121 gcgcatatga tcaaatttcg gggacacttc ctcatcgagg gggacctgaa cccagacaac
     3181 agcgatgtcg acaaactctt tatccaactg gttcagactt acaatcagct tttcgaagag
     3241 aacccgatca acgcatccgg agttgacgcc aaagcaatcc tgagcgctag gctgtccaaa
     3301 tcccggcggc tcgaaaacct catcgcacag ctccctgggg agaagaagaa cggcctgttt
     3361 ggtaatctta tcgccctgtc actcgggctg acccccaact ttaaatctaa cttcgacctg
     3421 gccgaagatg ccaagcttca actgagcaaa gacacctacg atgatgatct cgacaatctg
     3481 ctggcccaga tcggcgacca gtacgcagac ctttttttgg cggcaaagaa cctgtcagac
     3541 gccattctgc tgagtgatat tctgcgagtg aacacggaga tcaccaaagc tccgctgagc
     3601 gctagtatga tcaagcgcta tgatgagcac caccaagact tgactttgct gaaggccctt
     3661 gtcagacagc aactgcctga gaagtacaag gaaattttct tcgatcagtc taaaaatggc
     3721 tacgccggat acattgacgg cggagcaagc caggaggaat tttacaaatt tattaagccc
     3781 atcttggaaa aaatggacgg caccgaggag ctgctggtaa agcttaacag agaagatctg
     3841 ttgcgcaaac agcgcacttt cgacaatgga agcatccccc accagattca cctgggcgaa
     3901 ctgcacgcta tcctcaggcg gcaagaggat ttctacccct ttttgaaaga taacagggaa
     3961 aagattgaga aaatcctcac atttcggata ccctactatg taggccccct cgcccgggga
     4021 aattccagat tcgcgtggat gactcgcaaa tcagaagaga ccatcactcc ctggaacttc
     4081 gaggaagtcg tggataaggg ggcctctgcc cagtccttca tcgaaaggat gactaacttt
     4141 gataaaaatc tgcctaacga aaaggtgctt cctaaacact ctctgctgta cgagtacttc
     4201 acagtttata acgagctcac caaggtcaaa tacgtcacag aagggatgag aaagccagca
     4261 ttcctgtctg gagagcagaa gaaagctatc gtggacctcc tcttcaagac gaaccggaaa
     4321 gttaccgtga aacagctcaa agaagactat ttcaaaaaga ttgaatgttt cgactctgtt
     4381 gaaatcagcg gagtggagga tcgcttcaac gcatccctgg gaacgtatca cgatctcctg
     4441 aaaatcatta aagacaagga cttcctggac aatgaggaga acgaggacat tcttgaggac
     4501 attgtcctca cccttacgtt gtttgaagat agggagatga ttgaagaacg cttgaaaact
     4561 tacgctcatc tcttcgacga caaagtcatg aaacagctca agaggcgccg atatacagga
     4621 tgggggcggc tgtcaagaaa actgatcaat gggatccgag acaagcagag tggaaagaca
     4681 atcctggatt ttcttaagtc cgatggattt gccaaccgga acttcatgca gttgatccat
     4741 gatgactctc tcacctttaa ggaggacatc cagaaagcac aagtttctgg ccagggggac
     4801 agtcttcacg agcacatcgc taatcttgca ggtagcccag ctatcaaaaa gggaatactg
     4861 cagaccgtta aggtcgtgga tgaactcgtc aaagtaatgg gaaggcataa gcccgagaat
     4921 atcgttatcg agatggcccg agagaaccaa actacccaga agggacagaa gaacagtagg
     4981 gaaaggatga agaggattga agagggtata aaagaactgg ggtcccaaat ccttaaggaa
     5041 cacccagttg aaaacaccca gcttcagaat gagaagctct acctgtacta cctgcagaac
     5101 ggcagggaca tgtacgtgga tcaggaactg gacatcaatc ggctctccga ctacgacgtg
     5161 gatcatatcg tgccccagtc ttttctcaaa gatgattcta ttgataataa agtgttgaca
     5221 agatccgata aaaatagagg gaagagtgat aacgtcccct cagaagaagt tgtcaagaaa
     5281 atgaaaaatt attggcggca gctgctgaac gccaaactga tcacacaacg gaagttcgat
     5341 aatctgacta aggctgaacg aggtggcctg tctgagttgg ataaagccgg cttcatcaaa
     5401 aggcagcttg ttgagacacg ccagatcacc aagcacgtgg cccaaattct cgattcacgc
     5461 atgaacacca agtacgatga aaatgacaaa ctgattcgag aggtgaaagt tattactctg
     5521 aagtctaagc tggtctcaga tttcagaaag gactttcagt tttataaggt gagagagatc
     5581 aacaattacc accatgcgca tgatgcctac ctgaatgcag tggtaggcac tgcacttatc
     5641 aaaaaatatc ccaagcttga atctgaattt gtttacggag actataaagt gtacgatgtt
     5701 aggaaaatga tcgcaaagtc tgagcaggaa ataggcaagg ccaccgctaa gtacttcttt
     5761 tacagcaata ttatgaattt tttcaagacc gagattacac tggccaatgg agagattcgg
     5821 aagcgaccac ttatcgaaac aaacggagaa acaggagaaa tcgtgtggga caagggtagg
     5881 gatttcgcga cagtccggaa ggtcctgtcc atgccgcagg tgaacatcgt taaaaagacc
     5941 gaagtacaga ccggaggctt ctccaaggaa agtatcctcc cgaaaaggaa cagcgacaag
     6001 ctgatcgcac gcaaaaaaga ttgggacccc aagaaatacg gcggattcga ttctcctaca
     6061 gtcgcttaca gtgtactggt tgtggccaaa gtggagaaag ggaagtctaa aaaactcaaa
     6121 agcgtcaagg aactgctggg catcacaatc atggagcgat caagcttcga aaaaaacccc
     6181 atcgactttc tcgaggcgaa aggatataaa gaggtcaaaa aagacctcat cattaagctt
     6241 cccaagtact ctctctttga gcttgaaaac ggccggaaac gaatgctcgc tagtgcgggc
     6301 gagctgcaga aaggtaacga gctggcactg ccctctaaat acgttaattt cttgtatctg
     6361 gccagccact atgaaaagct caaagggtct cccgaagata atgagcagaa gcagctgttc
     6421 gtggaacaac acaaacacta ccttgatgag atcatcgagc aaataagcga attctccaaa
     6481 agagtgatcc tcgccgacgc taacctcgat aaggtgcttt ctgcttacaa taagcacagg
     6541 gataagccca tcagggagca ggcagaaaac attatccact tgtttactct gaccaacttg
     6601 ggcgcgcctg cagccttcaa gtacttcgac accaccatag acagaaagcg gtacacctct
     6661 acaaaggagg tcctggacgc cacactgatt catcagtcaa ttacggggct ctatgaaaca
     6721 agaatcgacc tctctcagct cggtggagac agcagggctg accccaagaa gaagaggaag
     6781 gtgaccggtg caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct
     6841 ggaccgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     6901 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     6961 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     7021 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     7081 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     7141 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     7201 gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg
     7261 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     7321 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     7381 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     7441 tgatgtacaa gtaactgcag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     7501 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     7561 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     7621 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     7681 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     7741 gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     7801 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     7861 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     7921 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     7981 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     8041 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     8101 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     8161 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     8221 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     8281 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     8341 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     8401 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     8461 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     8521 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     8581 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     8641 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     8701 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     8761 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     8821 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     8881 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     8941 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     9001 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     9061 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     9121 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     9181 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     9241 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     9301 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     9361 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     9421 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     9481 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     9541 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     9601 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     9661 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     9721 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     9781 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     9841 gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa
     9901 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     9961 aagctacaac aaggcaaggc ttgaccgac
//
