LOCUS       sequence-211319-        3921 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For rapid cloning and expression of sgRNAs in mammalian cells. 
            Contains copGFP for easy FACS sorting..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3921)
  TITLE     Minimal CRISPR guide (sgRNA) expression plasmid with copGFP and dual
            BbsI cloning sites
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3921)
  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: 
            "Unpublished"
COMMENT     Sequence Label: GuideVector_BbsI
FEATURES             Location/Qualifiers
     source          1..3921
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        96..475
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        476..679
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     630..650
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          771..903
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     CDS             939..1604
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     CDS             939..1604
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     primer_bind     complement(1618..1637)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    1647..1728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     1672..1691
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    1857..1881
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     1898..1914
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1898..1914
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        complement(1929..2004)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2023..2271)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2081..2100)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2251..2271)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2272..2288)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(2360..2948)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2440..2459)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3029..3823)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(3029..3823)
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3140..3159)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3750..3769
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 ataaatcaat attggctatt ggccattgca tacgttgtat ctatatcata atatgtacat
       61 ttatattggc tcatgtccaa tatgaccgcc atgttggcat tgattattga ctagttatta
      121 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      181 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      241 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      301 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtccgcc
      361 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      421 acgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
      481 gcggttttgg cagtacacca atgggcgtgg atagcggttt gactcacggg gatttccaag
      541 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
      601 aaaatgtcgt aataaccccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga
      661 ggtctatata agcagaggtc gtttagtgaa ccgtcagatc actagtagct ttattgcggt
      721 agtttatcac agttaaattg ctaacgcagt cagtgctcga ctgatcacag gtaagtatca
      781 aggttacaag acaggtttaa ggaggccaat agaaactggg cttgtcgaga cagagaagat
      841 tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc tttctctcca
      901 caggggtacc gaagccgcta gcgctaccgg tcgccaccat gcccgccatg aagatcgagt
      961 gccgcatcac cggcaccctg aacggcgtgg agttcgagct ggtgggcggc ggagagggca
     1021 cccccgagca gggccgcatg accaacaaga tgaagagcac caaaggcgcc ctgaccttca
     1081 gcccctacct gctgagccac gtgatgggct acggcttcta ccacttcggc acctacccca
     1141 gcggctacga gaaccccttc ctgcacgcca tcaacaacgg cggctacacc aacacccgca
     1201 tcgagaagta cgaggacggc ggcgtgctgc acgtgagctt cagctaccgc tacgaggccg
     1261 gccgcgtgat cggcgacttc aaggtggtgg gcaccggctt ccccgaggac agcgtgatct
     1321 tcaccgacaa gatcatccgc agcaacgcca ccgtggagca cctgcacccc atgggcgata
     1381 acgtgctggt gggcagcttc gcccgcacct tcagcctgcg cgacggcggc tactacagct
     1441 tcgtggtgga cagccacatg cacttcaaga gcgccatcca ccccagcatc ctgcagaacg
     1501 ggggccccat gttcgccttc cgccgcgtgg aggagctgca cagcaacacc gagctgggca
     1561 tcgtggagta ccagcacgcc ttcaagaccc ccatcgcctt cgccagatct cgagctcgat
     1621 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     1681 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     1741 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     1801 aacctctaca aatgtggtac ttaagagggg gagaccaaag ggcgagacgt taagggccaa
     1861 ctttgtacaa gaaagctgtt aattaatcgc gactagtcag gaaacagcta tgacggtcta
     1921 gaaaaaaagc accgactcgg tgccactttt tcaagttgat aacggactag ccttatttta
     1981 acttgctatt tctagctcta aaacaggtct tctcgaagac ccggtgtttc gtcctttcca
     2041 caagatatat aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc
     2101 cattttaaaa cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca
     2161 cgtattttgt actaatatct ttgtgtttac agtcaaatta attctaatta tctctctaac
     2221 agccttgtat cgtatatgca aatatgaagg aatcatggga aataggccct cactggccgt
     2281 cgttttactg atcagatatc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2341 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2401 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2461 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttacggga tacctgtccg
     2521 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2581 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2641 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2701 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2761 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2821 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2881 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2941 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3001 cacgttaagg gattttggtc atgccgtctc agaagaactc gtcaagaagg cgatagaagg
     3061 cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt
     3121 cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg
     3181 ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat
     3241 tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgctcgcct
     3301 tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct
     3361 gatcgacaag accggcttcc atccgagtac gtgctctctc gatgcgatgt ttcgcttggt
     3421 ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga
     3481 tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc
     3541 ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagtacagct gcgcaaggaa
     3601 cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ttgcagttca ttcagggcac
     3661 cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg
     3721 cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc
     3781 aagcggccgg agaacctgcg tgcaatccat cttgttcaat cataatatta ttgaagcatt
     3841 tatcagggtt cgtctcgtcc cggtctcctc ccatgcatgt caatattggc cattagccat
     3901 attattcatt ggttatatag c
//
