LOCUS       sequence-233628-        6516 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 6516)
  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 6516)
  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..6516
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     349..368
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    653..774
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(690..709)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     744..763
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(823..839)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(823..839)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(836..858)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    847..863
                     /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(871..877,878..895,896..901))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    916..937
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1054..1071)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1225..1810)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1305..1324)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1981..2772,2773..2841))
                     /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     2604..2623
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2842..2946)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3021..3040)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3147..3395
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3147..3167
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3318..3337
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        3585..3964
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3965..4168
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4119..4139
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4213..4232
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4213..4231
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4273..4282
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4279..4986
                     /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(4424..4442)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4692..4711
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4987..5013
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(5087..5104)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5093..5317
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5363..5791
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5450..5469)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5660..5681
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5800..5820)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5805..6134
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5985..6120
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6047..6066
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             join(6201..6516,1..479)
                     /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(6255..6274)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
       61 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
      121 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
      181 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
      241 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
      301 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
      361 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
      421 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
      481 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
      541 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
      601 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
      661 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
      721 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
      781 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
      841 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
      901 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
      961 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     1021 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     1081 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     1141 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     1201 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     1261 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     1321 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     1381 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     1441 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     1501 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     1561 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     1621 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     1681 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     1741 acaaaccacc gctggtagcg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     1801 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     1861 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     1921 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     1981 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     2041 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     2101 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     2161 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     2221 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     2281 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     2341 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     2401 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     2461 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     2521 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     2581 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     2641 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     2701 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     2761 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     2821 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     2881 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     2941 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
     3001 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
     3061 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
     3121 aggcaaggct tgaccgactt aattaagagg gcctatttcc catgattcct tcatatttgc
     3181 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     3241 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     3301 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     3361 tggctttata tatcttgtgg aaaggacgaa acaccggacc tatagggtca atgaatctgt
     3421 gcgtgtgcca taagtaatta aaaattaccc accacaggag cacctgaaaa caggtgcttg
     3481 gcacggagac gatatatcgt ctcttttttt caattgcatg aagaatctgc ttagggttag
     3541 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
     3601 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     3661 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     3721 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     3781 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     3841 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     3901 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     3961 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     4021 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     4081 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     4141 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
     4201 gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc gtttaaactt
     4261 aagcttgccg gtgccaccat ggtgagcaag ggcgaggagg ataacatggc catcatcaag
     4321 gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc
     4381 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
     4441 aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc
     4501 aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag
     4561 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag
     4621 gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc
     4681 ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg
     4741 atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac
     4801 ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg
     4861 cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc
     4921 atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg
     4981 tacaagtacc catacgatgt tccagattac gcttaagaat tctgcagata tccagcacag
     5041 tggcggccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct
     5101 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     5161 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     5221 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     5281 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     5341 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     5401 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     5461 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     5521 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     5581 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5641 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     5701 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     5761 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     5821 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     5881 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     5941 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     6001 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     6061 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     6121 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     6181 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     6241 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     6301 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     6361 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     6421 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     6481 tgggcgaagt gccggggcag gatctcctgt catctc
//
