LOCUS       sequence-314976-        4384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for a customizable guide RNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4384)
  AUTHORS   Pijnappel WWMP.
  TITLE     Pijnappel Lab Plasmids
  JOURNAL   Springer Nature (in press).
REFERENCE   2  (bases 1 to 4384)
  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: "Springer 
            Nature (in press)."
COMMENT     Sequence Label: pCRII-BbsI-sgRNAscaffold
FEATURES             Location/Qualifiers
     source          1..4384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        160..408
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     160..180
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     331..350
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        428..503
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(515..539)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(613..632)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(614..632)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(639..656)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(639..655)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(648..670)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      796..1224
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(883..902)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1093..1114
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             1568..2362
                     /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(1622..1641)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2232..2251
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(2380..2448,2449..3240)
                     /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(2598..2617)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3411..3999
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3900..3919
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4153..4170
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4287..4308
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4323..4328,4329..4346,4347..4353)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4361..4377
                     /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     join(4366..4384,1..4)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caggaaacag ctatgaccat gattacgcca agcttggtac cgagctcgga tccactagta
       61 acggccgcca gtgtgctgga attcgccctt tgtacaaaaa agcaggcttt aaaggaacca
      121 attcagtcga ctggatccgg taccaaggtc gggcaggaag agggcctatt tcccatgatt
      181 ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat taatttgact
      241 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
      301 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
      361 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg ggtcttcgag
      421 aagacctgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
      481 aaaaagtggc accgagtcgg tgcttttttt ctagacccag ctttcttgta caaagttggc
      541 attaaagccg aattctgcag atatccatca cactggcggc cgctcgagca tgcatctaga
      601 gggcccaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt tttacaacgt
      661 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
      721 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
      781 ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
      841 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
      901 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
      961 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     1021 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     1081 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     1141 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     1201 tttaacaaaa atttaacgcg aattttaaca aaattcaggg cgcaagggct gctaaaggaa
     1261 gcggaacacg tagaaagcca gtccgcagaa acggtgctga ccccggatga atgtcagcta
     1321 ctgggctatc tggacaaggg aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg
     1381 gcttacatgg cgatagctag actgggcggt tttatggaca gcaagcgaac cggaattgcc
     1441 agctggggcg ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt
     1501 gccgccaagg atctgatggc gcaggggatc aagatctgat caagagacag gatgaggatc
     1561 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     1621 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     1681 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     1741 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     1801 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     1861 ggggcaggat ctcctgtcat cccaccttgc tcctgccgag aaagtatcca tcatggctga
     1921 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     1981 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     2041 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     2101 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     2161 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     2221 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     2281 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     2341 ccttcttgac gagttcttct gaattgaaaa aggaagagta tgagtattca acatttccgt
     2401 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     2461 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     2521 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     2581 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     2641 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     2701 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     2761 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     2821 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     2881 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     2941 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3001 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     3061 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     3121 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     3181 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3241 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     3301 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3361 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3421 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3481 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3541 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     3601 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3661 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3721 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3781 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3841 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3901 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3961 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4021 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4081 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4141 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     4201 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     4261 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     4321 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     4381 caca
//
