LOCUS       sequence-239576-        7896 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human expression vector for planctomyces CasX and sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7896)
  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 7896)
  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 62.5
FEATURES             Location/Qualifiers
     source          1..7896
                     /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             4822..4842
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4843..4890
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             4900..4956
                     /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             4957..5553
                     /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     5450..5470
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5587..5604)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5593..5800
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5809..5949
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5809..5938
                     /label=AAV2 ITR
     rep_origin      6024..6479
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6111..6130)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6321..6342
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6496..6515)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6615..6637
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6675..6693)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6761..6865
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6866..6934,6935..7726)
                     /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(7084..7103)
                     /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 gcggggcggg gsggggsgrg gggsggggsg gggsgrggcg
     1501 gagaggtgcg 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 catgcaagag atcaagagaa tcaacaagat cagaaggaga
     1921 ctggtcaagg acagcaacac aaagaaggcc ggcaagacag gccccatgaa aaccctgctc
     1981 gtcagagtga tgacccctga cctgagagag cggctggaaa acctgagaaa gaagcccgag
     2041 aacatccctc agcctatcag caacaccagc agggccaacc tgaacaagct gctgaccgac
     2101 tacaccgaga tgaagaaagc catcctgcac gtgtactggg aagagttcca gaaagacccc
     2161 gtgggcctga tgagcagagt tgctcagccc gctcctaaga acatcgacca gagaaagctg
     2221 atccccgtga aggacggcaa cgagagactg acctctagcg gctttgcctg cagccagtgt
     2281 tgccagcctc tgtacgtgta caagctggaa caagtgaacg acaagggcaa gccccacacc
     2341 aactacttcg gcagatgcaa cgtgtccgag cacgagaggc tgatcctgct gtctcctcac
     2401 aagcccgagg ccaacgatga gctggtcaca tacagcctgg gcaagttcgg acagagagcc
     2461 ctggacttct acagcatcca cgtgaccagg gagagcaatc accctgtgaa gcccctggaa
     2521 cagatcggcg gcaatagctg tgcctctgga cctgtgggaa aagccctgag cgacgcctgt
     2581 atgggagccg tggcatcctt cctgaccaag taccaggaca tcatcctgga acaccagaaa
     2641 gtgatcaaga agaacgagaa aagactggcc aacctcaagg atatcgccag cgctaacggc
     2701 ctggcctttc ctaagatcac cctgcctcca cagcctcaca ccaaagaggg catcgaggcc
     2761 tacaacaacg tggtggccca gatcgtgatt tgggtcaacc tgaatctgtg gcagaagctg
     2821 aagatcggca gggacgaagc caagccactg cagagactga agggcttccc tagcttccct
     2881 ctggtggaaa gacaggccaa tgaagtggat tggtgggaca tggtctgcaa cgtgaagaag
     2941 ctgatcaacg agaagaaaga ggatggcaag gttttctggc agaacctggc cggctacaag
     3001 agacaagaag ccctgctgcc ttacctgagc agcgaagagg accggaagaa gggcaagaag
     3061 ttcgccagat accagttcgg cgacctgctg ctgcacctgg aaaagaagca cggcgaggac
     3121 tggggcaaag tgtacgatga ggcctgggag agaatcgaca agaaggtgga aggcctgagc
     3181 aagcacatta agctggaaga ggaaagaagg agcgaggacg cccaatctaa agccgctctg
     3241 accgattggc tgagagccaa ggccagcttt gtgatcgagg gcctgaaaga ggccgacaag
     3301 gacgagttct gcagatgcga gctgaagctg cagaagtggt acggcgatct gagaggcaag
     3361 cccttcgcca ttgaggccga gaacagcatc ctggacatca gcggcttcag caagcagtac
     3421 aactgcgcct tcatttggca gaaagacggc gtcaagaaac tgaacctgta cctgatcatc
     3481 aattacttca aaggcggcaa gctgcggttc aagaagatca aacccgaggc cttcgaggct
     3541 aacagattct acaccgtgat caacaaaaag tccggcgaga tcgtgcccat ggaagtgaac
     3601 ttcaacttcg acgaccccaa cctgattatc ctgcctctgg ccttcggcaa gagacagggc
     3661 agagagttca tctggaacga tctgctgagc ctggaaaccg gctctctgaa gctggccaat
     3721 ggcagagtga tcgagaaaac cctgtacaac aggagaacca gacaggacga gcctgctctg
     3781 tttgtggccc tgaccttcga gagaagagag gtgctggaca gcagcaacat caagcccatg
     3841 aacctgatcg gcatcgaccg gggcgagaat atccctgctg tgatcgccct gacagaccct
     3901 gaaggatgcc cactgagcag attcaaggac tccctgggca accctacaca catcctgaga
     3961 atcggcgaga gctacaaaga gaagcagagg acaatccagg ccgccaaaga ggtggaacag
     4021 agaagagccg gcggatactc taggaagtac gccagcaagg ccaagaatct ggccgacgac
     4081 atggtccgaa acaccgccag agatctgctg tactacgccg tgacacagga cgccatgctg
     4141 atcttcgaga atctgagcag aggcttcggc cggcagggca agagaacctt tatggccgag
     4201 aggcagtaca ccagaatgga agattggctc acagctaaac tggcctacga gggactgccc
     4261 agcaagacct acctgtccaa aacactggcc cagtatacct ccaagacctg cagcaattgc
     4321 ggcttcacca tcaccagcgc cgactacgac agagtgctgg aaaagctcaa gaaaaccgcc
     4381 accggctgga tgaccaccat caacggcaaa gagctgaagg ttgagggcca gatcacctac
     4441 tacaacaggt acaagaggca gaacgtcgtg aaggatctga gcgtggaact ggacagactg
     4501 agcgaagaga gcgtgaacaa cgacatcagc agctggacaa agggcagatc aggcgaggct
     4561 ctgagcctgc tgaagaagag gtttagccac agacctgtgc aagagaagtt cgtgtgcctg
     4621 aactgcggct tcgagacaca cgccgatgaa caggctgccc tgaacattgc cagaagctgg
     4681 ctgttcctga gaagccaaga gtacaagaag taccagacca acaagaccac cggcaacacc
     4741 gacaagaggg cctttgtgga aacctggcag agcttctaca gaaaaaagct gaaagaagtc
     4801 tggaagcccg ccgtgactag tccaaaaaag aagagaaagg tagattacaa agatgacgat
     4861 gacaaagact acaaggatga tgatgataag ggatccggcg caacaaactt ctctctgctg
     4921 aaacaagccg gagatgtcga agagaatcct ggaccgaccg agtacaagcc cacggtgcgc
     4981 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     5041 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     5101 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     5161 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     5221 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     5281 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga
     5341 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     5401 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     5461 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     5521 acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagaattcct agagctcgct
     5581 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     5641 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     5701 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     5761 agggggagga ttgggaagag aatagcaggc atgctgggga gcggccgcag gaacccctag
     5821 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     5881 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     5941 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     6001 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     6061 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     6121 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     6181 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     6241 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     6301 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     6361 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     6421 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     6481 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     6541 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     6601 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     6661 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     6721 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     6781 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     6841 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     6901 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     6961 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     7021 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     7081 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     7141 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     7201 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     7261 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     7321 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     7381 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     7441 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     7501 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     7561 atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa
     7621 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     7681 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     7741 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     7801 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     7861 agcgtcagac cccgtagaaa agatcaaagg atcttc
//
