LOCUS       40924_21903        4199 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector PGK1p-Csy4-pA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4199)
  AUTHORS   Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK.
  TITLE     Multiplexed and programmable regulation of gene networks with an 
            integrated RNA and CRISPR/Cas toolkit in human cells
  JOURNAL   Mol. Cell 54 (4), 698-710 (2014)
  PUBMED    24837679
REFERENCE   2  (bases 1 to 4199)
  AUTHORS   Perli SD.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-MAY-2014) Electrical Engineering and Computer Science,
            Massachusetts Institute of Technology, 77, Massachusetts Avenue, 
            Cambridge, MA 02139, USA
REFERENCE   3  (bases 1 to 4199)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 4199)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol. Cell";
            date: "2014"; volume: "54"; issue: "4"; pages: "698-710"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (04-MAY-2014) Electrical Engineering and Computer Science, 
            Massachusetts Institute of Technology, 77, Massachusetts Avenue, 
            Cambridge, MA 02139, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4199
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      72..527
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        660..1159
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     CDS             1201..1218
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             1219..1239
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     CDS             1252..1809
                     /codon_start=1
                     /label=Csy4
                     /note="CRISPR-associated endoribonuclease responsible for 
                     pre-crRNA processing in Pseudomonas aeruginosa (Haurwitz et
                     al., 2010)"
                     /translation="DHYLDIRLRPDPEFPPAQLMSVLFGKLHQALVAQGGDRIGVSFPD
                     LDESRSRLGERLRIHASADDLRALLARPWLEGLRDHLQFGEPAVVPHPTPYRQVSRVQA
                     KSNPERLRRRLMRRHDLSEEEARKRIPDTVARALDLPFVTLRSQSTGQHFRLFIRHGPL
                     QVTAEEGGFTCYGLSKGGFVPWF"
     polyA_signal    complement(1985..2106)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(2520..3108)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3282..4139)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(join(4140..4199,1..45))
                     /label=AmpR promoter
ORIGIN
        1 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
       61 agtgccacct gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
      121 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      181 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      241 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      301 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      361 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      421 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      481 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttccc attcgccatt
      541 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagcc
      601 caagctacca tgataagtaa gtaatattaa ggtacgggag gtacttggag gtacctaccg
      661 ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg
      721 cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg
      781 ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag
      841 gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg
      901 tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg
      961 gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg
     1021 tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc
     1081 cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc
     1141 ctcatctccg ggcctttcga cctgcatccc aagcttacgc gtgccaccat gaaatcttct
     1201 caccatcacc atcaccatga aaacctgtac ttccaatcca atgcagctag cgaccactat
     1261 ctggacatca gactgaggcc cgatcctgag ttccctcccg cccagctgat gagcgtgctg
     1321 tttggcaagc tgcatcaggc tctggtcgcc caaggcggag acagaatcgg cgtgtccttc
     1381 cccgacctgg acgagtcccg gagtcgcctg ggcgagcggc tgagaatcca cgccagcgca
     1441 gacgatctgc gcgccctgct ggcccggcct tggctggagg gcctgcggga tcatctgcag
     1501 tttggcgagc ccgccgtggt gccacaccca acaccctacc gccaggtgag ccgcgtgcag
     1561 gccaagtcaa atcccgagag actgcggcgg aggctgatga ggcgacatga tctgagcgag
     1621 gaggaggcca gaaagagaat ccccgacaca gtggccagag ccctggatct gccatttgtg
     1681 accctgcgga gccagagcac tggccagcat ttcagactgt tcatcagaca cgggcccctg
     1741 caggtgacag ccgaggaggg cggatttaca tgctatggcc tgtctaaagg cggcttcgtg
     1801 ccctggttct gatgagagct ctaagcggcc gcataagata tcagccatgg cttcccgccg
     1861 gcggtggcgg cgcaggatga tggcacgctg cccatgtctt gtgcccagga gagcgggatg
     1921 gaccgtcacc ctgcagcctg tgcttctgct aggatcaatg tgtaggctag tcgagcagac
     1981 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     2041 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     2101 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
     2161 gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tcgagtatgt cgaccgatgc
     2221 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg
     2281 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct
     2341 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2401 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2461 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2521 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2581 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2641 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2701 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2761 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     2821 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     2881 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     2941 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3001 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3061 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3121 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3181 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3241 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3301 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3361 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3421 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3481 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3541 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3601 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3661 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3721 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3781 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3841 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3901 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3961 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4021 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4081 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4141 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggata
//
