LOCUS       sequence-189203-        4434 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid expresses a gRNA targeting the first exon of human B2M.
            Most efficient gRNA targeting B2M, leading to ablation of B2M and 
            MHC class I surface expression in 50% of transfected cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4434)
  AUTHORS   Mandal PK, Ferreira LM, Collins R, Meissner TB, Boutwell CL, Friesen
            M, Vrbanac V, Garrison BS, Stortchevoi A, Bryder D, Musunuru K, 
            Brand H, Tager AM, Allen TM, Talkowski ME, Rossi DJ, Cowan CA
  TITLE     Efficient ablation of genes in human hematopoietic stem and effector
            cells using CRISPR/Cas9.
  JOURNAL   Cell Stem Cell. 2014 Nov 6;15(5):643-52. doi: 
            10.1016/j.stem.2014.10.004. Epub 2014 Nov 6.
  PUBMED    25517468
REFERENCE   2  (bases 1 to 4434)
  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"; doi: 
            "10.1016/j.stem.2014.10.004"; journalName: "Cell Stem Cell"; date: 
            "2014-11-6- 6"; volume: "15"; issue: "5"; pages: "643-52"
COMMENT     Sequence Label: B2M Bulldozer (gRNA crB2M_13)
FEATURES             Location/Qualifiers
     source          1..4434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        171..419
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     171..191
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     342..361
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        440..515
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(527..551)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(629..648)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(630..648)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(655..672)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(655..671)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(664..686)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      812..1240
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(899..918)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1109..1130
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             1584..2378
                     /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(1638..1657)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2248..2267
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(2396..2464,2465..3256)
                     /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     complement(2614..2633)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3427..4015
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3916..3935
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4169..4186
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4303..4324
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4339..4344,4345..4362,4363..4369)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4377..4393
                     /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)."
     primer_bind     4382..4404
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4401..4417
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4401..4417
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 atttaggtga cactatagaa tactcaagct atgcatcaag cttggtaccg agctcggatc
       61 cactagtaac ggccgccagt gtgctggaat tcgcccttca ttgtacaaaa aagcaggctt
      121 taaaggaacc aattcagtcg actggatccg gtaccaaggt cgggcaggaa gagggcctat
      181 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa
      241 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
      301 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
      361 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
      421 ctactctctc tttctggccg ttttagagct agaaatagca agttaaaata aggctagtcc
      481 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctagaccc agctttcttg
      541 tacaaagttg gcattaatga agggcgaatt ctgcagatat ccatcacact ggcggccgct
      601 cgagcatgca tctagagggc ccaattcgcc ctatagtgag tcgtattaca attcactggc
      661 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
      721 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
      781 ccaacagttg cgcagcctga atggcgaatg gacgcgccct gtagcggcgc attaagcgcg
      841 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
      901 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
      961 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1021 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     1081 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     1141 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     1201 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat tcagggcgca
     1261 agggctgcta aaggaagcgg aacacgtaga aagccagtcc gcagaaacgg tgctgacccc
     1321 ggatgaatgt cagctactgg gctatctgga caagggaaaa cgcaagcgca aagagaaagc
     1381 aggtagcttg cagtgggctt acatggcgat agctagactg ggcggtttta tggacagcaa
     1441 gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa
     1501 actggatggc tttcttgccg ccaaggatct gatggcgcag gggatcaaga tctgatcaag
     1561 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     1621 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     1681 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     1741 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     1801 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     1861 tattgggcga agtgccgggg caggatctcc tgtcatccca ccttgctcct gccgagaaag
     1921 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     1981 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     2041 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     2101 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     2161 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     2221 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     2281 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     2341 gcatcgcctt ctatcgcctt cttgacgagt tcttctgaat tgaaaaagga agagtatgag
     2401 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2461 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2521 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     2581 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     2641 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2701 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2761 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2821 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2881 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     2941 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     3001 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3061 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     3121 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3181 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3241 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3301 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3361 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3421 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3481 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3541 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     3601 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3661 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3721 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3781 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3841 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3901 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3961 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4021 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4081 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4141 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4201 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     4261 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     4321 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     4381 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agct
//
