LOCUS       sequence-233627-        6525 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA for BhCas12b in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6525)
  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 6525)
  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: pZ148-BhCas12b-sgRNA-scaffold
FEATURES             Location/Qualifiers
     source          1..6525
                     /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        3594..3973
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3974..4177
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4128..4148
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4222..4241
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4222..4240
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4282..4291
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4288..4995
                     /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(4433..4451)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4701..4720
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4996..5022
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(5096..5113)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5102..5326
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5372..5800
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5459..5478)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5669..5690
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5809..5829)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5814..6143
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5994..6129
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6056..6075
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             join(6210..6525,1..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(6264..6283)
                     /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 acaccggttc tgtcttttgg tcaggacaac
     3421 cgtctagcta taagtgctgc agggtgtgag aaactcctat tgctggacga tgtctcttac
     3481 gaggcattag cacggagacg atatatcgtc tctttttttc aattgcatga agaatctgct
     3541 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
     3601 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     3661 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     3721 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     3781 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     3841 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     3901 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     3961 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     4021 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     4081 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     4141 taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac
     4201 tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagcg
     4261 tttaaactta agcttgccgg tgccaccatg gtgagcaagg gcgaggagga taacatggcc
     4321 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
     4381 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     4441 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
     4501 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     4561 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     4621 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     4681 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     4741 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     4801 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     4861 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     4921 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     4981 gacgagctgt acaagtaccc atacgatgtt ccagattacg cttaagaatt ctgcagatat
     5041 ccagcacagt ggcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg
     5101 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     5161 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     5221 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     5281 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     5341 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     5401 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     5461 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     5521 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     5581 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     5641 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     5701 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     5761 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     5821 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     5881 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     5941 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     6001 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     6061 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     6121 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     6181 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     6241 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     6301 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     6361 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     6421 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     6481 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctc
//
