LOCUS       sequence-239575-        7917 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human expression vector for deltaproteobacteria CasX and sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7917)
  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 7917)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2019 Feb 4. pii: 10.1038/s41586-019-0908-x. doi: 
            10.1038/s41586-019-0908-x."
COMMENT     Sequence Label: pBLO 62.4
FEATURES             Location/Qualifiers
     source          1..7917
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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..899
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     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..1834
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1846..1855
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1858..1878
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4843..4863
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4864..4911
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             4921..4977
                     /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             4978..5574
                     /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     5471..5491
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5608..5625)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5614..5821
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5830..5970
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5830..5959
                     /label=AAV2 ITR
     rep_origin      6045..6500
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6132..6151)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6342..6363
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6517..6536)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6636..6658
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6696..6714)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6782..6886
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6887..6955,6956..7747)
                     /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(7105..7124)
                     /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 atactgttct 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 gcggggsggg gsggggsgrg gggsggggsg gggsgrggcg
     1501 gagaggkgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
     1561 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
     1621 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     1681 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
     1741 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta
     1801 cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catggcccca
     1861 aagaagaagc ggaaggtcag cgaaaagaga atcaacaaga tcaggaagaa gctgagcgcc
     1921 gacaacgcca ccaagcctgt gtctaggagt ggccccatga aaaccctgct cgtcagagtg
     1981 atgaccgacg acctgaagaa gagactggaa aagaggagga agaaacccga agtgatgccc
     2041 caagtgatca gcaacaacgc cgccaacaac ctgagaatgc tgctggacga ctacaccaag
     2101 atgaaggaag ccatcctgca ggtctactgg caagagttca aggacgacca cgtgggcctg
     2161 atgtgcaagt ttgcccagcc tgccagcaag aagatcgacc agaacaagct gaagcccgag
     2221 atggacgaga agggcaatct gaccacagcc ggctttgcct gctctcagtg tggccagcct
     2281 ctgttcgtgt acaagctgga acaggtgtcc gagaaaggca aggcctacac caactacttc
     2341 ggcagatgta acgtggccga gcacgagaag ctgattctgc tggcccagct gaaacctgag
     2401 aaggactctg atgaggccgt gacctacagc ctgggcaagt ttggacagag agccctggac
     2461 ttctacagca tccacgtgac caaagaaagc acacaccccg tgaagcccct ggctcagatc
     2521 gccggcaata gatacgcctc tggacctgtg ggcaaagccc tgtccgatgc ctgcatggga
     2581 acaatcgcca gcttcctgag caagtaccag gacatcatca tcgagcacca gaaggtggtc
     2641 aagggcaacc agaagagact ggaaagcctg agggagctgg ccggcaaaga gaacctggaa
     2701 taccccagcg tgaccctgcc tcctcagcct cacacaaaag aaggcgtgga cgcctacaac
     2761 gaagtgatcg ccagagtgag aatgtgggtc aacctgaacc tgtggcagaa gctgaaactg
     2821 tccagggacg acgccaagcc tctgctgaga ctgaagggct tccctagctt ccccgtggtg
     2881 gaaagaaggg agaatgaggt ggactggtgg aacaccatca acgaagtcaa gaaactgatc
     2941 gatgccaaga gagacatggg cagagtgttt tggagcggag tgaccgccga gaagagaaac
     3001 accatcctgg aaggctacaa ctacctgcct aacgagaacg accacaagaa gagagagggc
     3061 agcctggaaa accctaagaa gcccgccaag agacagttcg gcgacctgct gctgtacctg
     3121 gagaagaagt atgccggcga ctggggcaaa gtgttcgatg aggcctggga gagaatcgac
     3181 aagaagattg ccggcctgac cagccacatc gagagagagg aagccagaaa cgccgaggac
     3241 gcccagtcta aagccgtgct gacagattgg ctgagagcca aggccagctt cgtgctggaa
     3301 aggctgaaag aaatggatga gaaagagttc tacgcctgcg agatccagct gcagaagtgg
     3361 tacggggacc tgagaggcaa tccctttgcc gtggaagccg agaacagagt ggtggacatc
     3421 agcggcttta gcatcggctc tgacggccac tccatccagt acaggaacct gctggcctgg
     3481 aagtacctgg aaaatggcaa gagagagttc tacctgctga tgaactacgg caagaagggc
     3541 agaatcagat tcaccgacgg caccgacatc aagaaaagcg gcaagtggca gggcctgctg
     3601 tatggcggcg gaaaggccaa agtgatcgac ctgaccttcg atcccgacga cgagcagctg
     3661 atcattctgc ctctggcctt cggcaccaga cagggcagag agttcatctg gaacgatctg
     3721 ctgtccctgg aaaccggcct gatcaagctg gccaacggca gggtgatcga gaaaaccatc
     3781 tacaacaaga aaatcggcag agatgagccc gcactgtttg tggccctgac ctttgagagg
     3841 agagaagtgg tcgatcccag caacatcaag cccgtgaacc tgatcggagt ggacagaggc
     3901 gagaatatcc ctgccgtgat tgccctgaca gaccctgagg gatgtcccct gcctgagttt
     3961 aaggatagct ctggcggccc taccgacatc ctgagaatcg gcgagggcta caaagagaag
     4021 cagagagcca tccaggccgc caaagaggtg gaacagagaa gagccggcgg atacagcaga
     4081 aagttcgcca gcaagtccag aaacctggcc gatgacatgg tcaggaacag cgccagggac
     4141 ctgttctacc acgccgtgac acatgatgcc gtgctggtgt tcgagaacct gtcaagaggc
     4201 ttcggcagac agggaaaaag aaccttcatg accgagagac agtacacgaa gatggaagat
     4261 tggctcaccg ccaaactggc ctacgagggc ctgacaagca agacctacct gtccaagaca
     4321 ctggcccagt acacctccaa gacctgctcc aattgcggct tcaccatcac caccgccgac
     4381 tacgatggaa tgctcgtgag actgaagaaa acctccgacg gctgggccac cacactgaac
     4441 aacaaagagc tgaaagccga gggccagatc acctactaca acagatacaa gaggcagacc
     4501 gtcgagaaag agctgtccgc cgaactcgac agactgagcg aggaatccgg caacaacgac
     4561 atcagcaagt ggaccaaggg acgcagagat gaggccctgt tcctgctgaa gaagaggttc
     4621 agccacagac ctgtgcaaga gcagtttgtg tgcctggatt gcggccacga agtgcacgca
     4681 gatgaacagg ccgctctgaa tatcgccaga agctggctgt tcctgaacag caacagcacc
     4741 gagttcaaga gctacaagag cggcaagcag cctttcgttg gagcctggca ggccttctac
     4801 aagagaaggc tcaaagaagt gtggaagccc aacgccacta gtccaaaaaa gaagagaaag
     4861 gtagattaca aagatgacga tgacaaagac tacaaggatg atgatgataa gggatccggc
     4921 gcaacaaact tctctctgct gaaacaagcc ggagatgtcg aagagaatcc tggaccgacc
     4981 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     5041 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     5101 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     5161 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     5221 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     5281 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     5341 tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     5401 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     5461 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     5521 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaacgcgt
     5581 taagaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     5641 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     5701 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     5761 gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg
     5821 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     5881 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     5941 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     6001 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     6061 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     6121 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     6181 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     6241 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     6301 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     6361 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     6421 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     6481 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     6541 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     6601 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     6661 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     6721 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     6781 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     6841 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     6901 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     6961 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     7021 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     7081 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     7141 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     7201 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     7261 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     7321 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     7381 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     7441 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     7501 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     7561 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc
     7621 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     7681 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     7741 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     7801 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     7861 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
