LOCUS      pZ148-BhCas12b-sgRNA-scaffold        6528 bp    DNA     circular SYN 14-APR-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 6528)
  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 6528)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            //gt12365.cn
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..6528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        207..586
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        587..790
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     741..761
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     835..854
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        835..853
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      895..904
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             901..1608
                     /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(1046..1064)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1314..1333
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1609..1635
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1709..1726)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1715..1939
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1985..2413
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2072..2091)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2282..2303
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2422..2442)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2427..2756
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2607..2742
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2669..2688
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2823..3617
                     /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(2877..2896)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3487..3506
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3791..3912
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3828..3847)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3882..3901
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3961..3977)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3961..3977)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3974..3996)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3985..4001
                     /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(4009..4015,4016..4033,4034..4039))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4054..4075
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4192..4209)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4363..4951)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4443..4462)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5122..5913,5914..5982))
                     /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     5745..5764
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5983..6087)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6162..6181)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6288..6528,1..8)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6288..6308
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6459..6478
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gaaacaccgg ttctgtcttt tggtcaggac aaccgtctag ctataagtgc tgcagggtgt
       61 gagaaactcc tattgctgga cgatgtctct tacgaggcat tagcacggag acgatatatc
      121 gtctcttttt ttcaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
      181 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
      241 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      301 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      361 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      421 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      481 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      541 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      601 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      661 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      721 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      781 aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac
      841 gactcactat agggagaccc aagctggcta gcgtttaaac ttaagcttgc cggtgccacc
      901 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
      961 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     1021 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     1081 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     1141 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     1201 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     1261 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     1321 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     1381 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     1441 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     1501 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     1561 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta cccatacgat
     1621 gttccagatt acgcttaaga attctgcaga tatccagcac agtggcggcc gctcgagtct
     1681 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     1741 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     1801 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     1861 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     1921 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     1981 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2041 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2101 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2161 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2221 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2281 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2341 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2401 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     2461 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     2521 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2581 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     2641 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     2701 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     2761 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
     2821 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     2881 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     2941 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     3001 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     3061 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     3121 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     3181 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     3241 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     3301 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     3361 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     3421 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     3481 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     3541 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     3601 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     3661 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     3721 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     3781 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3841 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3901 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     3961 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4021 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4081 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4141 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4201 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4261 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4321 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4381 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4441 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4501 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4561 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4621 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4681 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4741 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4801 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4861 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4921 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4981 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5041 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5101 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5161 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5221 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5281 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5341 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5401 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5461 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5521 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5581 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5641 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5701 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5761 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5821 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5881 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5941 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6001 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6061 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     6121 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     6181 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     6241 agcaaaattt aagctacaac aaggcaaggc ttgaccgact taattaagag ggcctatttc
     6301 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
     6361 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     6421 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     6481 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggac
//