LOCUS       Exported                7165 bp ds-DNA     circular SYN 20-JUN-2018
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    1255_pAAV-U6-SA-BbsI-MluI-gRNA-CB-SACas9-HA-OLLAS-spA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7165)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Jun 20, 2018 from SnapGene 2.8.3
            http://www.snapgene.com
COMMENT     Component Fragments
            #1: FRAGMENT of pZAC2.1rhEPO.36.Q-L
             parent position: from 1309 to 239
             original length: 3894
             molecule position: from 1 to 3894
            Left Terminus
              XhoI site #1
            Right Terminus
              BglII site #1
            #2: FRAGMENT of pAAVNEWTBG-BI-aGAL
             parent position: from 5114 to 316
             original length: 896
             molecule position: from 3895 to 4790
            Left Terminus
              BamHI site #1
            Right Terminus
              XhoI site #1
            http://www.informaxinc.com/
FEATURES             Location/Qualifiers
     source          1..7165
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    1..185
                     /label=ITR
                     /note="5'ITR"
     misc_feature    complement(1..173)
                     /label=ITR
                     /note="3'ITR"
     promoter        255..495
                     /label=U6 Promoter
                     /note="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    276..310
                     /note="U6ForSeq Primer"
     misc_feature    504..525
                     /note="NEW CLONING_SITE_FOR_GRNA"
     misc_feature    524..525
                     /note="nt leftover from previous site"
     misc_feature    526..606
                     /note="sgRNA scaffold"
     misc_feature    526..539
                     /note="crRNA Direct Repeat Portion +1 to +14"
     misc_feature    540..543
                     /note="synthetic tetraloop"
     misc_feature    544..606
                     /note="TracrRNA"
     misc_feature    607
                     /note="Additional ""T"" introduced in gblock"
     misc_feature    608..629
                     /note="Cloning Site for Additional Guide RNA cassettes"
     misc_feature    630..1078
                     /note="Promoter cassette from pAAV-CB-EGFP"
     enhancer        685..810
                     /note="Partial CMV enhancer"
     misc_feature    complement(698..719)
                     /note="CBP Rev3 Primer"
     promoter        811..1031
                     /note="CB promoter"
     misc_feature    905..906
                     /note="5 bp deletion between these two bases"
     TATA_signal     1007..1014
                     /note="TATA"
     primer_bind     complement(1053..1079)
                     /note="CLONE_CB_R_NcoI"
     CDS             1088..1108
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     misc_feature    1133..4288
                     /note="hSaCas9"
     misc_feature    1532..1534
                     /note="Ub high (AAA = WT, AGA = deUb)"
     misc_feature    1532..1534
                     /note="K151R"
     misc_feature    3335..3337
                     /note="K752R"
     CDS             4289..4336
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4343..4369
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /note="HA"
                     /translation="YPYDVPDYA"
     misc_feature    4370..4411
                     /note="OLLAS tag"
     polyA_signal    4427..4484
                     /note="Syn PolyA"
     misc_feature    4485..4657
                     /label=ITR
                     /note="3'ITR"
     misc_feature    complement(4682..7097)
                     /label=pEMBL8 Plasmid Backbone
                     /note="pEMBL8 Plasmid Backbone"
     rep_origin      complement(4891..5479)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5650..6510)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVVMVTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6511..6615)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      complement(6642..7097)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
       61 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
      121 actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc
      181 tctggtgtac aaaaaagcag gctttaaagg aaccaattca gtcgactgga tccggtacca
      241 aggtcgggca ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca
      301 aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa
      361 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
      421 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
      481 tcttgtggaa aggacgaaac accgggtctt cacgcgtgaa gaccagtttt agtactctgg
      541 aaacagaatc tactaaaaca aggcaaaatg ccgtgtttat ctcgtcaact tgttggcgag
      601 atttttttct agacccagct ttcaagcttg attaacccgc catgctactt atctacgtag
      661 ccatgctcta ggaagatcgg aattcgccct taagctagta tgccaagtac gccccctatt
      721 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      781 tttcctactt ggcagtacat ctactcgagg ccacgttctg cttcactctc cccatctccc
      841 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
      901 gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc
      961 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
     1021 cgcgcggcgg gcgggagcgg gatcagaatg atctgatatc atcgatgaat tcgagctcac
     1081 catggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccaagcggaa
     1141 ctacatcctg ggcctggaca tcggcatcac cagcgtgggc tacggcatca tcgactacga
     1201 gacacgggac gtgatcgatg ccggcgtgcg gctgttcaaa gaggccaacg tggaaaacaa
     1261 cgagggcagg cggagcaaga gaggcgccag aaggctgaag cggcggaggc ggcatagaat
     1321 ccagagagtg aagaagctgc tgttcgacta caacctgctg accgaccaca gcgagctgag
     1381 cggcatcaac ccctacgagg ccagagtgaa gggcctgagc cagaagctga gcgaggaaga
     1441 gttctctgcc gccctgctgc acctggccaa gagaagaggc gtgcacaacg tgaacgaggt
     1501 ggaagaggac accggcaacg agctgtccac cagagagcag atcagccgga acagcaaggc
     1561 cctggaagag aaatacgtgg ccgaactgca gctggaacgg ctgaagaaag acggcgaagt
     1621 gcggggcagc atcaacagat tcaagaccag cgactacgtg aaagaagcca aacagctgct
     1681 gaaggtgcag aaggcctacc accagctgga ccagagcttc atcgacacct acatcgacct
     1741 gctggaaacc cggcggacct actatgaggg acctggcgag ggcagcccct tcggctggaa
     1801 ggacatcaaa gaatggtacg agatgctgat gggccactgc acctacttcc ccgaggaact
     1861 gcggagcgtg aagtacgcct acaacgccga cctgtacaac gccctgaacg acctgaacaa
     1921 tctcgtgatc accagggacg agaacgagaa gctggaatat tacgagaagt tccagatcat
     1981 cgagaacgtg ttcaagcaga agaagaagcc caccctgaag cagatcgcca aagaaatcct
     2041 cgtgaacgaa gaggatatta agggctacag agtgaccagc accggcaagc ccgagttcac
     2101 caacctgaag gtgtaccacg acatcaagga cattaccgcc cggaaagaga ttattgagaa
     2161 cgccgagctg ctggatcaga ttgccaagat cctgaccatc taccagagca gcgaggacat
     2221 ccaggaagaa ctgaccaatc tgaactccga gctgacccag gaagagatcg agcagatctc
     2281 taatctgaag ggctataccg gcacccacaa cctgagcctg aaggccatca acctgatcct
     2341 ggacgagctg tggcacacca acgacaacca gatcgctatc ttcaaccggc tgaagctggt
     2401 gcccaagaag gtggacctgt cccagcagaa agagatcccc accaccctgg tggacgactt
     2461 catcctgagc cccgtcgtga agagaagctt catccagagc atcaaagtga tcaacgccat
     2521 catcaagaag tacggcctgc ccaacgacat cattatcgag ctggcccgcg agaagaactc
     2581 caaggacgcc cagaaaatga tcaacgagat gcagaagcgg aaccggcaga ccaacgagcg
     2641 gatcgaggaa atcatccgga ccaccggcaa agagaacgcc aagtacctga tcgagaagat
     2701 caagctgcac gacatgcagg aaggcaagtg cctgtacagc ctggaagcca tccctctgga
     2761 agatctgctg aacaacccct tcaactatga ggtggaccac atcatcccca gaagcgtgtc
     2821 cttcgacaac agcttcaaca acaaggtgct cgtgaagcag gaagaaaaca gcaagaaggg
     2881 caaccggacc ccattccagt acctgagcag cagcgacagc aagatcagct acgaaacctt
     2941 caagaagcac atcctgaatc tggccaaggg caagggcaga atcagcaaga ccaagaaaga
     3001 gtatctgctg gaagaacggg acatcaacag gttctccgtg cagaaagact tcatcaaccg
     3061 gaacctggtg gataccagat acgccaccag aggcctgatg aacctgctgc ggagctactt
     3121 cagagtgaac aacctggacg tgaaagtgaa gtccatcaat ggcggcttca ccagctttct
     3181 gcggcggaag tggaagttta agaaagagcg gaacaagggg tacaagcacc acgccgagga
     3241 cgccctgatc attgccaacg ccgatttcat cttcaaagag tggaagaaac tggacaaggc
     3301 caaaaaagtg atggaaaacc agatgttcga ggaaaggcag gccgagagca tgcccgagat
     3361 cgaaaccgag caggagtaca aagagatctt catcaccccc caccagatca agcacattaa
     3421 ggacttcaag gactacaagt acagccaccg ggtggacaag aagcctaata gagagctgat
     3481 taacgacacc ctgtactcca cccggaagga cgacaagggc aacaccctga tcgtgaacaa
     3541 tctgaacggc ctgtacgaca aggacaatga caagctgaaa aagctgatca acaagagccc
     3601 cgaaaagctg ctgatgtacc accacgaccc ccagacctac cagaaactga agctgattat
     3661 ggaacagtac ggcgacgaga agaatcccct gtacaagtac tacgaggaaa ccgggaacta
     3721 cctgaccaag tactccaaaa aggacaacgg ccccgtgatc aagaagatta agtattacgg
     3781 caacaaactg aacgcccatc tggacatcac cgacgactac cccaacagca gaaacaaggt
     3841 cgtgaagctg tccctgaagc cctacagatt cgacgtgtac ctggacaatg gcgtgtacaa
     3901 gttcgtgacc gtgaagaatc tggatgtgat caaaaaagaa aactactacg aagtgaatag
     3961 caagtgctat gaggaagcta agaagctgaa gaagatcagc aaccaggccg agtttatcgc
     4021 ctccttctac aacaacgatc tgatcaagat caacggcgag ctgtatagag tgatcggcgt
     4081 gaacaacgac ctgctgaacc ggatcgaagt gaacatgatc gacatcacct accgcgagta
     4141 cctggaaaac atgaacgaca agaggccccc caggatcatt aagacaatcg cctccaagac
     4201 ccagagcatt aagaagtaca gcacagacat tctgggcaac ctgtatgaag tgaaatctaa
     4261 gaagcaccct cagatcatca aaaagggcaa aaggccggcg gccacgaaaa aggccggcca
     4321 ggcaaaaaag aaaaagggat cctacccata cgatgttcca gattacgcta gcggcttcgc
     4381 caacgagctt ggacccaggt tgatgggaaa gtaagaattc ctagagcgaa taaaagatct
     4441 ttattttcat tagatctgtg tgttggtttt ttgtgtgatg cagctacgta gataagtagc
     4501 atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc
     4561 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     4621 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcat taatgaatcg gccaacgcgc
     4681 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4741 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4801 cacagaatca ggggataacg caggaaagaa catgtgagca aaaccgcagc aaaaggccag
     4861 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4921 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4981 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5041 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5101 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5161 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5221 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5281 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     5341 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5401 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5461 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5521 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5581 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5641 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5701 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5761 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5821 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5881 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5941 tttgcgcaac gttgttacca ttactacagg catcgtggtg tcacgctcgt cgtttggtat
     6001 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6061 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6121 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6181 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6241 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6301 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccact
     6361 attgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6421 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6481 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6541 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6601 aataggggtt ccgcgcacat ttccccgaaa gatgccacct gaaattataa acgttaatat
     6661 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     6721 aatcggaaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     6781 agtttggaac aagagtccac tattgaggaa cgtgaactcc agcgtcaaag ggcgaaaaac
     6841 cgtctatcgg ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     6901 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     6961 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     7021 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     7081 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc
     7141 ggtgcgggcc tcttcgctat tacgc
//
