LOCUS       sequence-256596-        4435 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA for SpCas9 mediated B2M KO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4435)
  TITLE     Lian Lab CRISPR plasmids
REFERENCE   2  (bases 1 to 4435)
  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"
COMMENT     Sequence Label: pLR06_pGuide_B2M_sgRNA2
FEATURES             Location/Qualifiers
     source          1..4435
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..28)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /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     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(1819..2613)
                     /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(1930..1949)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2540..2559
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      complement(2957..3385)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3067..3088)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3279..3298
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3511..3533
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3525..3542
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3526..3542
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3549..3568
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3549..3567
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3710..3958
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3710..3730
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3881..3900
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3980..4055
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(4067..4091)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(4179..4197)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4180..4197)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4215..4231)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4215..4231)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4228..4250)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4239..4255
                     /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(4263..4269,4270..4287,4288..4293))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4308..4329
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaattc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg
     1861 cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag
     1921 ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag
     1981 ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca
     2041 ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc
     2101 gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag
     2161 accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg
     2221 gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt
     2281 ctcggcagga gcaaggtggg atgacaggag atcctgcccc ggcacttcgc ccaatagcag
     2341 ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt
     2401 ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc
     2461 ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga
     2521 gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg
     2581 agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg
     2641 atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac
     2701 tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc
     2761 tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt
     2821 tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatccgggg
     2881 tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc agcccttgcg
     2941 ccctgaattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3001 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3061 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     3121 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     3181 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     3241 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     3301 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     3361 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     3421 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     3481 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     3541 gtgaattgta atacgactca ctatagggcg aattgggccc tctagatgca tgctcgagcg
     3601 gccgccagtg tgatggatat ctgcagaatt cgcccttcat tgtacaaaaa agcaggcttt
     3661 aaaggaacca attcagtcga ctggatccgg taccaaggtc gggcaggaag agggcctatt
     3721 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat
     3781 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     3841 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     3901 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga
     3961 caaagtcaca tggttcacag ttttagagct agaaatagca agttaaaata aggctagtcc
     4021 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctagaccc agctttcttg
     4081 tacaaagttg gcattaatga agggcgaatt ccagcacact ggcggccgtt actagtggat
     4141 ccgagctcgg taccaagctt gatgcatagc ttgagtattc tatagtgtca cctaaatagc
     4201 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4261 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4321 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4381 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgct
//
