LOCUS       sequence-236177-        6519 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA for BvCas12b in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6519)
  AUTHORS   Strecker J, Jones S, Koopal B, Schmid-Burgk J, Zetsche B, Gao L, 
            Makarova KS, Koonin EV, Zhang F
  TITLE     Engineering of CRISPR-Cas12b for human genome editing.
  JOURNAL   Nat Commun. 2019 Jan 22;10(1):212. doi: 10.1038/s41467-018-08224-4.
  PUBMED    30670702
REFERENCE   2  (bases 1 to 6519)
  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: "Nat 
            Commun."; date: "2019-01-22"; volume: "10(1):212. doi"; pages: " 
            10.1038/s41467-018-08224-4"
COMMENT     Sequence Label: pZ147-BvCas12b-sgRNA-scaffold
FEATURES             Location/Qualifiers
     source          1..6519
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        198..577
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        578..781
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     732..752
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     826..845
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        826..844
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      886..895
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             892..1599
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1037..1055)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1305..1324
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1600..1626
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1700..1717)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1706..1930
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1976..2404
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2063..2082)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2273..2294
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2413..2433)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2418..2747
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2598..2733
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2660..2679
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2814..3608
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2868..2887)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3478..3497
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3782..3903
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3819..3838)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3873..3892
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3952..3968)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3952..3968)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3965..3987)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3976..3992
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4000..4006,4007..4024,4025..4030))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4045..4066
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4183..4200)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4354..4942)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4434..4453)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5113..5904,5905..5973))
                     /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     5736..5755
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5974..6078)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6153..6172)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6279..6519,1..8)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6279..6299
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6450..6469
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gaaacaccgg acctataggg tcaatgaatc tgtgcgtgtg ccataagtaa ttaaaaatta
       61 cccaccacag gagcacctga aaacaggtgc ttggcacgga gacgatatat cgtctctttt
      121 tttcaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
      181 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
      241 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      301 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      361 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      421 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      481 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      541 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
      601 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      661 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
      721 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      781 tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta
      841 tagggagacc caagctggct agcgtttaaa cttaagcttg ccggtgccac catggtgagc
      901 aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg
      961 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     1021 gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg
     1081 gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac
     1141 atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     1201 ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc
     1261 atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag
     1321 aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag
     1381 ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag
     1441 accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag
     1501 ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag
     1561 ggccgccact ccaccggcgg catggacgag ctgtacaagt acccatacga tgttccagat
     1621 tacgcttaag aattctgcag atatccagca cagtggcggc cgctcgagtc tagagggccc
     1681 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     1741 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     1801 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     1861 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     1921 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     1981 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2041 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2101 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2161 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2221 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2281 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2341 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2401 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     2461 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     2521 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     2581 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     2641 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     2701 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     2761 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     2821 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     2881 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     2941 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     3001 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     3061 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3121 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     3181 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     3241 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     3301 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     3361 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     3421 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     3481 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     3541 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     3601 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     3661 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     3721 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     3781 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     3841 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     3901 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     3961 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4021 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4081 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4141 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4201 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4261 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4321 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4381 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4441 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4501 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4561 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4621 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4681 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4741 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4801 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4861 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4921 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4981 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5041 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5101 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     5161 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     5221 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     5281 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     5341 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     5401 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     5461 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5521 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5581 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     5641 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5701 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5761 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5821 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5881 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     5941 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6001 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6061 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     6121 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     6181 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
     6241 taagctacaa caaggcaagg cttgaccgac ttaattaaga gggcctattt cccatgattc
     6301 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     6361 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     6421 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     6481 atttcgattt cttggcttta tatatcttgt ggaaaggac
//
