LOCUS       40924_29581        3192 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pm!sp-gg, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3192)
  AUTHORS   Chavez A, Pruitt BW, Tuttle M, Shapiro RS, Cecchi RJ, Winston J, 
            Turczyk BM, Tung M, Collins JJ, Church GM.
  TITLE     Precise Cas9 targeting enables genomic mutation prevention
  JOURNAL   Proc. Natl. Acad. Sci. U.S.A. (2018) In press
  PUBMED    29555762
REFERENCE   2  (bases 1 to 3192)
  AUTHORS   Chavez A, Pruitt BW, Tuttle M, Shapiro RS, Cecchi RJ, Winston J, 
            Turczyk BM, Tung M, Collins JJ, Church GM.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-MAR-2018) Synthetic Biology, Wyss Institute, 3 
            Blackfan Circle, Floor 5, Boston, MA 02115, USA
REFERENCE   3  (bases 1 to 3192)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 3192)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc. Natl.
            Acad. Sci. U.S.A. (2018) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (02-MAR-2018) Synthetic Biology, Wyss Institute, 3 Blackfan Circle, 
            Floor 5, Boston, MA 02115, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3192
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..19
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    26..44
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    67..87
                     /label=golden gate cassette
                     /note="golden gate cassette"
     misc_feature    complement(67..73)
                     /label=SapI
                     /note="SapI"
     misc_feature    74..80
                     /label=padding
                     /note="padding"
     misc_feature    81..87
                     /label=SapI
                     /note="SapI"
     repeat_region   88..123
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes
                     CRISPR/Cas system"
     CDS             complement(988..1518)
                     /codon_start=1
                     /label=GmR
                     /note="gentamycin acetyltransferase"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(1707..1735)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      complement(2379..2924)
                     /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 tccctatcag tgatagagat tgacatccct atcagtgata gagatactct gctcgcaagg
       61 agggttgaag agcattaatg gctcttcgtt ttagagctat gctgttttga atggtcccaa
      121 aaccgccgct atggtcccac gtagagcata cggaaaaaaa agtcaaaagc ctccgaccgg
      181 aggtcggcct tacttgctag caccatccag ctgcagctct ggcccgtgtc tcaaaatctc
      241 tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac
      301 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga
      361 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
      421 caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg
      481 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
      541 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
      601 tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct
      661 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
      721 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca
      781 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
      841 gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc
      901 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aggccgggaa
      961 gccgatctcg gcttgaacga attgttaggt ggcggtactt gggtcgatat caaagtgcat
     1021 cacttcttcc cgtatgccca actttgtata gagagccact gcgggatcgt caccgtaatc
     1081 tgcttgcacg tagatcacat aagcaccaag cgcgttggcc tcatgcttga ggagattgat
     1141 gagcgcggtg gcaatgccct gcctccggtg ctcgccggag actgcgagat catagatata
     1201 gatctcacta cgcggctgct caaacctggg cagaacgtaa gccgcgagag cgccaacaac
     1261 cgcttcttgg tcgaaggcag caagcgcgat gaatgtctta ctacggagca agttcccgag
     1321 gtaatcggag tccggctgat gttgggagta ggtggctacg tctccgaact cacgaccgaa
     1381 aagatcaaga gcagcccgca tggatttgac ttggtcaggg ccgagcctac atgtgcgaat
     1441 gatgcccata cttgagccac ctaactttgt tttagggcga ctgccctgct gcgtaacatc
     1501 gttgctgctg cgtaacatcg ttgctgctcc ataacatcaa acatcgaccc acggcgtaac
     1561 gcgcttgctg cttggatgcc cgaggcatag actgtacaaa aaaacagtca taacaagcca
     1621 tgaaaaccgc cactgcgccg ttaccaccgc tgcgttcggt caaggttctg gaccagttgc
     1681 gtgagcgcat acgctacttg cattacagtt tacgaaccga acaggcttat gtcaactggg
     1741 ttcgtgcctt catccgtttc cacggtgtgc gtcacccggc aaccttgggc agcagcgaag
     1801 tcgaggcatt tctgtcctgg ctggcgaacg agcgcaaggt ttcggtctcc acgcatcgtc
     1861 aggcattggc ggccttgctg ttcttctacg gcaaggtgct gtgcacggat ctgccctggc
     1921 ttcaggagat cggaagacct cggccgtcgc ggcgcttgcc tggcagttcc ctactctcgc
     1981 atggggagac cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg
     2041 gtcaggtggg accaccgcgc tactgccgcc aggcaaattc tgttttatca gaccgcttct
     2101 gcgttctgat ttaatctgta tcaggctgaa aatcttctct catccgccaa aacagccctc
     2161 gggacggatc gcttcatgtg gcaggagaaa aaaggctgca ccggtgcgtc agcagaatat
     2221 gtgatacagg atatattccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact
     2281 gcggcgagcg gaaatggctt acgaacgggg cggagatttc ctggaagatg ccaggaagat
     2341 acttaacagg gaagtgagag ggccgcggca aagccgtttt tccataggct ccgcccccct
     2401 gacaagcatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa
     2461 agataccagg cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg
     2521 tttaccggtg tcattccgct gttatggccg cgtttgtctc attccacgcc tgacactcag
     2581 ttccgggtag gcagttcgct ccaagctgga ctgtatgcac gaaccccccg ttcagtccga
     2641 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggaaagac atgcaaaagc
     2701 accactggca gcagccactg gtaattgatt tagaggagtt agtcttgaag tcatgcgccg
     2761 gttaaggcta aactgaaagg acaagttttg gtgactgcgc tcctccaagc cagttacctc
     2821 ggttcaaaga gttggtagct cagagaacct tcgaaaaacc gccctgcaag gcggtttttt
     2881 cgttttcaga gcaagagatt acgcgcagac caaaacgatc tcaagaagat catcttatta
     2941 aggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3001 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3061 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3121 cagcggaacc gctctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca
     3181 gcccggcgca ac
//
