LOCUS       sequence-235004-        7897 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 7897)
  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 7897)
  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..7897
                     /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..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
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1859..1879
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4823..4843
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4844..4891
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             4901..4957
                     /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             4958..5554
                     /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     5451..5471
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5588..5605)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5594..5801
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5810..5950
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5810..5939
                     /label=AAV2 ITR
     rep_origin      6025..6480
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6112..6131)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6322..6343
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6497..6516)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6616..6638
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6676..6694)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6762..6866
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6867..6935,6936..7727)
                     /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(7085..7104)
                     /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 gcatgcaaga gatcaagaga atcaacaaga tcagaaggag
     1921 actggtcaag gacagcaaca caaagaaggc cggcaagaca ggccccatga aaaccctgct
     1981 cgtcagagtg atgacccctg acctgagaga gcggctggaa aacctgagaa agaagcccga
     2041 gaacatccct cagcctatca gcaacaccag cagggccaac ctgaacaagc tgctgaccga
     2101 ctacaccgag atgaagaaag ccatcctgca cgtgtactgg gaagagttcc agaaagaccc
     2161 cgtgggcctg atgagcagag ttgctcagcc cgctcctaag aacatcgacc agagaaagct
     2221 gatccccgtg aaggacggca acgagagact gacctctagc ggctttgcct gcagccagtg
     2281 ttgccagcct ctgtacgtgt acaagctgga acaagtgaac gacaagggca agccccacac
     2341 caactacttc ggcagatgca acgtgtccga gcacgagagg ctgatcctgc tgtctcctca
     2401 caagcccgag gccaacgatg agctggtcac atacagcctg ggcaagttcg gacagagagc
     2461 cctggacttc tacagcatcc acgtgaccag ggagagcaat caccctgtga agcccctgga
     2521 acagatcggc ggcaatagct gtgcctctgg acctgtggga aaagccctga gcgacgcctg
     2581 tatgggagcc gtggcatcct tcctgaccaa gtaccaggac atcatcctgg aacaccagaa
     2641 agtgatcaag aagaacgaga aaagactggc caacctcaag gatatcgcca gcgctaacgg
     2701 cctggccttt cctaagatca ccctgcctcc acagcctcac accaaagagg gcatcgaggc
     2761 ctacaacaac gtggtggccc agatcgtgat ttgggtcaac ctgaatctgt ggcagaagct
     2821 gaagatcggc agggacgaag ccaagccact gcagagactg aagggcttcc ctagcttccc
     2881 tctggtggaa agacaggcca atgaagtgga ttggtgggac atggtctgca acgtgaagaa
     2941 gctgatcaac gagaagaaag aggatggcaa ggttttctgg cagaacctgg ccggctacaa
     3001 gagacaagaa gccctgctgc cttacctgag cagcgaagag gaccggaaga agggcaagaa
     3061 gttcgccaga taccagttcg gcgacctgct gctgcacctg gaaaagaagc acggcgagga
     3121 ctggggcaaa gtgtacgatg aggcctggga gagaatcgac aagaaggtgg aaggcctgag
     3181 caagcacatt aagctggaag aggaaagaag gagcgaggac gcccaatcta aagccgctct
     3241 gaccgattgg ctgagagcca aggccagctt tgtgatcgag ggcctgaaag aggccgacaa
     3301 ggacgagttc tgcagatgcg agctgaagct gcagaagtgg tacggcgatc tgagaggcaa
     3361 gcccttcgcc attgaggccg agaacagcat cctggacatc agcggcttca gcaagcagta
     3421 caactgcgcc ttcatttggc agaaagacgg cgtcaagaaa ctgaacctgt acctgatcat
     3481 caattacttc aaaggcggca agctgcggtt caagaagatc aaacccgagg ccttcgaggc
     3541 taacagattc tacaccgtga tcaacaaaaa gtccggcgag atcgtgccca tggaagtgaa
     3601 cttcaacttc gacgacccca acctgattat cctgcctctg gccttcggca agagacaggg
     3661 cagagagttc atctggaacg atctgctgag cctggaaacc ggctctctga agctggccaa
     3721 tggcagagtg atcgagaaaa ccctgtacaa caggagaacc agacaggacg agcctgctct
     3781 gtttgtggcc ctgaccttcg agagaagaga ggtgctggac agcagcaaca tcaagcccat
     3841 gaacctgatc ggcatcgacc ggggcgagaa tatccctgct gtgatcgccc tgacagaccc
     3901 tgaaggatgc ccactgagca gattcaagga ctccctgggc aaccctacac acatcctgag
     3961 aatcggcgag agctacaaag agaagcagag gacaatccag gccgccaaag aggtggaaca
     4021 gagaagagcc ggcggatact ctaggaagta cgccagcaag gccaagaatc tggccgacga
     4081 catggtccga aacaccgcca gagatctgct gtactacgcc gtgacacagg acgccatgct
     4141 gatcttcgag aatctgagca gaggcttcgg ccggcagggc aagagaacct ttatggccga
     4201 gaggcagtac accagaatgg aagattggct cacagctaaa ctggcctacg agggactgcc
     4261 cagcaagacc tacctgtcca aaacactggc ccagtatacc tccaagacct gcagcaattg
     4321 cggcttcacc atcaccagcg ccgactacga cagagtgctg gaaaagctca agaaaaccgc
     4381 caccggctgg atgaccacca tcaacggcaa agagctgaag gttgagggcc agatcaccta
     4441 ctacaacagg tacaagaggc agaacgtcgt gaaggatctg agcgtggaac tggacagact
     4501 gagcgaagag agcgtgaaca acgacatcag cagctggaca aagggcagat caggcgaggc
     4561 tctgagcctg ctgaagaaga ggtttagcca cagacctgtg caagagaagt tcgtgtgcct
     4621 gaactgcggc ttcgagacac acgccgatga acaggctgcc ctgaacattg ccagaagctg
     4681 gctgttcctg agaagccaag agtacaagaa gtaccagacc aacaagacca ccggcaacac
     4741 cgacaagagg gcctttgtgg aaacctggca gagcttctac agaaaaaagc tgaaagaagt
     4801 ctggaagccc gccgtgacta gtccaaaaaa gaagagaaag gtagattaca aagatgacga
     4861 tgacaaagac tacaaggatg atgatgataa gggatccggc gcaacaaact tctctctgct
     4921 gaaacaagcc ggagatgtcg aagagaatcc tggaccgacc gagtacaagc ccacggtgcg
     4981 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga
     5041 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct
     5101 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     5161 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     5221 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     5281 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     5341 agtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     5401 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     5461 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     5521 cacctggtgc atgacccgca agcccggtgc ctgaacgcgt taagaattcc tagagctcgc
     5581 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     5641 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     5701 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     5761 aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta
     5821 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     5881 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     5941 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     6001 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     6061 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     6121 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     6181 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     6241 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     6301 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     6361 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     6421 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     6481 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     6541 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     6601 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     6661 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     6721 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     6781 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     6841 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     6901 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     6961 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     7021 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     7081 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     7141 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     7201 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     7261 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     7321 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     7381 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     7441 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     7501 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     7561 aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta
     7621 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     7681 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     7741 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     7801 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     7861 gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
