pZ149-pcDNA3-BhCas12b_v4

LOCUS       pZ149-pcDNA3-BhCas12b_v4                8912 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses BhCas12b_v4 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pZ149-pcDNA3-BhCas12b_v4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8912)
  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 8912)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..8912
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      683..692
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             695..715
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4061..4108
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4115..4195
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4269..4286)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4275..4499
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4545..4973
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4632..4651)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4842..4863
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4982..5002)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4987..5316
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5167..5302
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5229..5248
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5383..6177
                     /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(5437..5456)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6047..6066
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6351..6472
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6388..6407)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6442..6461
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6521..6537)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6521..6537)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6534..6556)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6545..6561
                     /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(6569..6599)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6614..6635
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6752..6769)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6923..7511)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7003..7022)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7682..8542)
                     /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     8305..8324
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8543..8647)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8722..8741)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacttaagc ttgccaccat ggccccaaag aagaagcgga aggtcggtat
      721 ccacggagtc ccagcagccg ccaccagatc cttcatcctg aagatcgagc ccaacgagga
      781 agtgaagaaa ggcctctgga aaacccacga ggtgctgaac cacggaatcg cctactacat
      841 gaatatcctg aagctgatcc ggcaagaggc catctacgag caccacgagc aggaccccaa
      901 gaatcccaag aaggtgtcca aggccgagat ccaggccgag ctgtgggatt tcgtgctgaa
      961 gatgcagaag tgcaacagct tcacacacga ggtggacaag gacgaggtgt tcaacatcct
     1021 gagagagctg tacgaggaac tggtgcccag cagcgtggaa aagaagggcg aagccaacca
     1081 gctgagcaac aagtttctgt accctctggt ggaccccaac agccagtctg gaaagggaac
     1141 agccagcagc ggcagaaagc ccagatggta caacctgaag attgccggcg atccctcctg
     1201 ggaagaagag aagaagaagt gggaagaaga taagaaaaag gacccgctgg ccaagatcct
     1261 gggcaagctg gctgagtacg gactgatccc tctgttcatc ccctacaccg acagcaacga
     1321 gcccatcgtg aaagaaatca agtggatgga aaagtcccgg aaccagagcg tgcggcggct
     1381 ggataaggac atgttcattc aggccctgga acggttcctg agctgggaga gctggaacct
     1441 gaaagtgaaa gaggaatacg agaaggtcga gaaagagtac aagaccctgg aagagaggat
     1501 caaagaggac atccaggctc tgaaggctct ggaacagtat gagaaagagc ggcaagaaca
     1561 gctgctgcgg gacaccctga acaccaacga gtaccggctg agcaagagag gccttagagg
     1621 ctggcgggaa atcatccaga aatggctgaa aatggacgag aacgagccct ccgagaagta
     1681 cctggaagtg ttcaaggact accagcggaa gcaccctaga gaggccggcg attacagcgt
     1741 gtacgagttc ctgtccaaga aagagaacca cttcatctgg cggaatcacc ctgagtaccc
     1801 ctacctgtac gccaccttct gcgagatcga caagaaaaag aaggacgcca agcagcaggc
     1861 caccttcaca ctggccgatc ctatcaatca ccctctgtgg gtccgattcg aggaaagaag
     1921 cggcagcaac ctgaacaagt acagaatcct gaccgagcag ctgcacaccg agaagctgaa
     1981 gaaaaagctg acagtgcagc tggaccggct gatctaccct acagaatctg gcggctggga
     2041 agagaagggc aaagtggaca ttgtgctgct gcccagccgg cagttctaca accagatctt
     2101 cctggacatc gaggaaaagg gcaagcacgc cttcacctac aaggatgaga gcatcaagtt
     2161 ccctctgaag ggcacactcg gcggagccag agtgcagttc gacagagatc acctgagaag
     2221 ataccctcac aaggtggaaa gcggcaacgt gggcagaatc tacttcaaca tgaccgtgaa
     2281 catcgagcct acagagtccc cagtgtccaa gtctctgaag atccaccggg acgacttccc
     2341 caaggtggtc aacttcaagc ccaaagaact gaccgagtgg atcaaggaca gcaagggcaa
     2401 gaaactgaag tccggcatcg agtccctgga aatcggcctg agagtgatga gcatcgacct
     2461 gggacagaga caggccgctg ccgcctctat tttcgaggtg gtggatcaga agcccgacat
     2521 cgaaggcaag ctgtttttcc caatcaaggg caccgagctg tatgccgtgc acagagccag
     2581 cttcaacatc aagctgcccg gcgagacact ggtcaagagc agagaagtgc tgcggaaggc
     2641 cagagaggac aatctgaaac tgatgaacca gaagctcaac ttcctgcgga acgtgctgca
     2701 cttccagcag ttcgaggaca tcaccgagag agagaagcgg gtcaccaagt ggatcagcag
     2761 acaagagaac agcgacgtgc ccctggtgta ccaggatgag ctgatccaga tccgcgagct
     2821 gatgtacaag ccttacaagg actgggtcgc cttcctgaag cagctccaca agagactgga
     2881 agtcgagatc ggcaaagaag tgaagcactg gcggaagtcc ctgagcgacg gaagaaaggg
     2941 cctgtacggc atctccctga agaacatcga cgagatcgat cggacccgga agttcctgct
     3001 gagatggtcc ctgaggccta ccgaacctgg cgaagtgcgt agactggaac ccggccagag
     3061 attcgccatc gaccagctga atcacctgaa cgccctgaaa gaagatcggc tgaagaagat
     3121 ggccaacacc atcatcatgc acgccctggg ctactgctac gacgtgcgga agaagaaatg
     3181 gcaggctaag aaccccgcct gccagatcat cctgttcgag gatctgagca actacaaccc
     3241 ctacggagaa aggtcccgct tcgagaacag ccggctcatg aagtggtcca gacgcgagat
     3301 ccccagacag gttgcactgc agggcgagat ctatggcctg caagtgggag aagtgggcgc
     3361 tcagttcagc agcagattcc acgccaagac aggcagccct ggcatcagat gtcgggtcgt
     3421 gaccaaagag aagctgcagg acaatcggtt cttcaagaat ctgcagagag agggcagact
     3481 gaccctggac aaaatcgccg tgctgaaaga gggcgatctg tacccagaca aaggcggcga
     3541 gaagttcatc agcctgagca aggatcggaa gtgcgtgacc acacacgccg acatcaacgc
     3601 cgctcagaac ctgcagaagc ggttctggac aagaacccac ggcttctaca aggtgtactg
     3661 caaggcctac caggtggacg gccagaccgt gtacatccct gagagcaagg accagaagca
     3721 gaagatcatc gaagagttcg gcgagggcta cttcattctg aaggacgggg tgtacgaatg
     3781 ggtcaacgcc ggcaagctga aaatcaagaa gggcagctcc aagcagagca gcagcgagct
     3841 ggtggatagc gacatcctga aagacagctt cgacctggcc tccgagctga aaggcgaaaa
     3901 gctgatgctg tacagggacc ccagcggcaa tgtgttcccc agcgacaaat ggatggccgc
     3961 tggcgtgttc ttcggaaagc tggaacgcat cctgatcagc aagctgacca accagtactc
     4021 catcagcacc atcgaggacg acagcagcaa gcagtctatg aaaaggccgg cggccacgaa
     4081 aaaggccggc caggcaaaaa agaaaaaggg atcctaccca tacgatgttc cagattacgc
     4141 ttatccctac gacgtgcctg attatgcata cccatatgat gtccccgact atgcctaaga
     4201 attctgcaga tatccagcac agtggcggcc gctcgagtct agagggcccg tttaaacccg
     4261 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     4321 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     4381 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     4441 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     4501 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     4561 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4621 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4681 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4741 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4801 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4861 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     4921 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     4981 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     5041 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     5101 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     5161 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     5221 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     5281 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     5341 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     5401 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     5461 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     5521 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     5581 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     5641 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     5701 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     5761 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     5821 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     5881 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     5941 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     6001 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     6061 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     6121 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     6181 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     6241 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     6301 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     6361 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     6421 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     6481 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     6541 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     6601 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     6661 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     6721 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     6781 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     6841 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     6901 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     6961 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     7021 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     7081 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     7141 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     7201 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     7261 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     7321 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     7381 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     7441 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     7501 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     7561 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     7621 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     7681 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     7741 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     7801 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     7861 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     7921 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     7981 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     8041 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     8101 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     8161 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     8221 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     8281 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     8341 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     8401 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     8461 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     8521 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     8581 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     8641 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     8701 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     8761 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     8821 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
     8881 gcttcgcgat gtacgggcca gatatacgcg tt
//