LOCUS       Exported                7918 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Human expression vector for deltaproteobacteria CasX and sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    pBLO 62.4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7918)
  AUTHORS   Liu JJ, Orlova N, Oakes BL, Ma E, Spinner HB, Baney KLM, Chuck J, 
            Tan D, Knott GJ, Harrington LB, Al-Shayeb B, Wagner A, Brotzmann J, 
            Staahl BT, Taylor KL, Desmarais J, Nogales E, Doudna JA
  TITLE     CasX enzymes comprise a distinct family of RNA-guided genome 
            editors.
  JOURNAL   Nature. 2019 Feb 4. pii: 10.1038/s41586-019-0908-x. doi: 
            10.1038/s41586-019-0908-x.
  PUBMED    30718774
REFERENCE   2  (bases 1 to 7918)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7918
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        651..891
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     651..671
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     822..841
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        1042..1327
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1329..1606
                     /label=chicken beta-actin promoter
     intron          1607..1835
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1847..1856
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1859..1879
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4844..4864
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4865..4912
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             4922..4978
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4979..5575
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     5472..5492
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5609..5626)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5615..5822
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5831..5971
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5831..5960
                     /label=AAV2 ITR
     rep_origin      6046..6501
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6133..6152)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6343..6364
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6518..6537)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6637..6659
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6697..6715)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6783..6887
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6888..7748
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7106..7125)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt gagggcctat
      661 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
      721 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
      781 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
      841 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacacca
      901 catctggcgc gtttattcca ttactttgga gccagtccca gcgactatgt cgtatggacg
      961 aagcgcttat ttatcggaga gaaaccgata agtaaaacgc atcaaaggcg tgtccggcga
     1021 gggcgagttt tttttggtac ccgttacata acttacggta aatggcccgc ctggctgacc
     1081 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg
     1141 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     1201 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca
     1261 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     1321 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     1381 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
     1441 gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg
     1501 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
     1561 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac
     1621 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
     1681 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
     1741 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt
     1801 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggcccc
     1861 aaagaagaag cggaaggtca gcgaaaagag aatcaacaag atcaggaaga agctgagcgc
     1921 cgacaacgcc accaagcctg tgtctaggag tggccccatg aaaaccctgc tcgtcagagt
     1981 gatgaccgac gacctgaaga agagactgga aaagaggagg aagaaacccg aagtgatgcc
     2041 ccaagtgatc agcaacaacg ccgccaacaa cctgagaatg ctgctggacg actacaccaa
     2101 gatgaaggaa gccatcctgc aggtctactg gcaagagttc aaggacgacc acgtgggcct
     2161 gatgtgcaag tttgcccagc ctgccagcaa gaagatcgac cagaacaagc tgaagcccga
     2221 gatggacgag aagggcaatc tgaccacagc cggctttgcc tgctctcagt gtggccagcc
     2281 tctgttcgtg tacaagctgg aacaggtgtc cgagaaaggc aaggcctaca ccaactactt
     2341 cggcagatgt aacgtggccg agcacgagaa gctgattctg ctggcccagc tgaaacctga
     2401 gaaggactct gatgaggccg tgacctacag cctgggcaag tttggacaga gagccctgga
     2461 cttctacagc atccacgtga ccaaagaaag cacacacccc gtgaagcccc tggctcagat
     2521 cgccggcaat agatacgcct ctggacctgt gggcaaagcc ctgtccgatg cctgcatggg
     2581 aacaatcgcc agcttcctga gcaagtacca ggacatcatc atcgagcacc agaaggtggt
     2641 caagggcaac cagaagagac tggaaagcct gagggagctg gccggcaaag agaacctgga
     2701 ataccccagc gtgaccctgc ctcctcagcc tcacacaaaa gaaggcgtgg acgcctacaa
     2761 cgaagtgatc gccagagtga gaatgtgggt caacctgaac ctgtggcaga agctgaaact
     2821 gtccagggac gacgccaagc ctctgctgag actgaagggc ttccctagct tccccgtggt
     2881 ggaaagaagg gagaatgagg tggactggtg gaacaccatc aacgaagtca agaaactgat
     2941 cgatgccaag agagacatgg gcagagtgtt ttggagcgga gtgaccgccg agaagagaaa
     3001 caccatcctg gaaggctaca actacctgcc taacgagaac gaccacaaga agagagaggg
     3061 cagcctggaa aaccctaaga agcccgccaa gagacagttc ggcgacctgc tgctgtacct
     3121 ggagaagaag tatgccggcg actggggcaa agtgttcgat gaggcctggg agagaatcga
     3181 caagaagatt gccggcctga ccagccacat cgagagagag gaagccagaa acgccgagga
     3241 cgcccagtct aaagccgtgc tgacagattg gctgagagcc aaggccagct tcgtgctgga
     3301 aaggctgaaa gaaatggatg agaaagagtt ctacgcctgc gagatccagc tgcagaagtg
     3361 gtacggggac ctgagaggca atccctttgc cgtggaagcc gagaacagag tggtggacat
     3421 cagcggcttt agcatcggct ctgacggcca ctccatccag tacaggaacc tgctggcctg
     3481 gaagtacctg gaaaatggca agagagagtt ctacctgctg atgaactacg gcaagaaggg
     3541 cagaatcaga ttcaccgacg gcaccgacat caagaaaagc ggcaagtggc agggcctgct
     3601 gtatggcggc ggaaaggcca aagtgatcga cctgaccttc gatcccgacg acgagcagct
     3661 gatcattctg cctctggcct tcggcaccag acagggcaga gagttcatct ggaacgatct
     3721 gctgtccctg gaaaccggcc tgatcaagct ggccaacggc agggtgatcg agaaaaccat
     3781 ctacaacaag aaaatcggca gagatgagcc cgcactgttt gtggccctga cctttgagag
     3841 gagagaagtg gtcgatccca gcaacatcaa gcccgtgaac ctgatcggag tggacagagg
     3901 cgagaatatc cctgccgtga ttgccctgac agaccctgag ggatgtcccc tgcctgagtt
     3961 taaggatagc tctggcggcc ctaccgacat cctgagaatc ggcgagggct acaaagagaa
     4021 gcagagagcc atccaggccg ccaaagaggt ggaacagaga agagccggcg gatacagcag
     4081 aaagttcgcc agcaagtcca gaaacctggc cgatgacatg gtcaggaaca gcgccaggga
     4141 cctgttctac cacgccgtga cacatgatgc cgtgctggtg ttcgagaacc tgtcaagagg
     4201 cttcggcaga cagggaaaaa gaaccttcat gaccgagaga cagtacacga agatggaaga
     4261 ttggctcacc gccaaactgg cctacgaggg cctgacaagc aagacctacc tgtccaagac
     4321 actggcccag tacacctcca agacctgctc caattgcggc ttcaccatca ccaccgccga
     4381 ctacgatgga atgctcgtga gactgaagaa aacctccgac ggctgggcca ccacactgaa
     4441 caacaaagag ctgaaagccg agggccagat cacctactac aacagataca agaggcagac
     4501 cgtcgagaaa gagctgtccg ccgaactcga cagactgagc gaggaatccg gcaacaacga
     4561 catcagcaag tggaccaagg gacgcagaga tgaggccctg ttcctgctga agaagaggtt
     4621 cagccacaga cctgtgcaag agcagtttgt gtgcctggat tgcggccacg aagtgcacgc
     4681 agatgaacag gccgctctga atatcgccag aagctggctg ttcctgaaca gcaacagcac
     4741 cgagttcaag agctacaaga gcggcaagca gcctttcgtt ggagcctggc aggccttcta
     4801 caagagaagg ctcaaagaag tgtggaagcc caacgccact agtccaaaaa agaagagaaa
     4861 ggtagattac aaagatgacg atgacaaaga ctacaaggat gatgatgata agggatccgg
     4921 cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgac
     4981 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     5041 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     5101 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     5161 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     5221 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     5281 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     5341 gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     5401 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     5461 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     5521 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg
     5581 ttaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     5641 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     5701 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     5761 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg
     5821 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     5881 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     5941 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     6001 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     6061 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     6121 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     6181 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     6241 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     6301 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     6361 ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga
     6421 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     6481 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     6541 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     6601 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     6661 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     6721 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     6781 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     6841 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     6901 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     6961 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     7021 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     7081 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     7141 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     7201 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     7261 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     7321 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     7381 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     7441 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     7501 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     7561 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg
     7621 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     7681 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     7741 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     7801 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     7861 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
