LOCUS       sequence-208240-        3934 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 3934)
  TITLE     Minimal CRISPR guide (sgRNA) expression plasmid with copGFP and dual
            BbsI cloning sites
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3934)
  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            "Unpublished"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..3934
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        167..546
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        547..750
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     701..721
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          842..974
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     CDS             1010..1675
                     /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             1010..1675
                     /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(1689..1708)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    1718..1799
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     1743..1762
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    1941..1965
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     1982..1998
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1982..1998
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        complement(2013..2088)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2107..2355)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2165..2184)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2335..2355)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2356..2372)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(2444..3032)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2524..2543)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3113..3907)
                     /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(3113..3907)
                     /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(3224..3243)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3834..3853
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 cgtctcgtcc cggtctcctc ccatgcatgt caatattggc cattagccat attattcatt
       61 ggttatatag cataaatcaa tattggctat tggccattgc atacgttgta tctatatcat
      121 aatatgtaca tttatattgg ctcatgtcca atatgaccgc catgttggca ttgattattg
      181 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      241 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      301 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      361 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      421 ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      481 tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      541 accatggtga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg
      601 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      661 cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg cggtaggcgt
      721 gtacggtggg aggtctatat aagcagaggt cgtttagtga accgtcagat cactagtagc
      781 tttattgcgg tagtttatca cagttaaatt gctaacgcag tcagtgctcg actgatcaca
      841 ggtaagtatc aaggttacaa gacaggttta aggaggccaa tagaaactgg gcttgtcgag
      901 acagagaaga ttcttgcgtt tctgataggc acctattggt cttactgaca tccactttgc
      961 ctttctctcc acaggggtac cgaagccgct agcgctaccg gtcgccacca tgcccgccat
     1021 gaagatcgag tgccgcatca ccggcaccct gaacggcgtg gagttcgagc tggtgggcgg
     1081 cggagagggc acccccgagc agggccgcat gaccaacaag atgaagagca ccaaaggcgc
     1141 cctgaccttc agcccctacc tgctgagcca cgtgatgggc tacggcttct accacttcgg
     1201 cacctacccc agcggctacg agaacccctt cctgcacgcc atcaacaacg gcggctacac
     1261 caacacccgc atcgagaagt acgaggacgg cggcgtgctg cacgtgagct tcagctaccg
     1321 ctacgaggcc ggccgcgtga tcggcgactt caaggtggtg ggcaccggct tccccgagga
     1381 cagcgtgatc ttcaccgaca agatcatccg cagcaacgcc accgtggagc acctgcaccc
     1441 catgggcgat aacgtgctgg tgggcagctt cgcccgcacc ttcagcctgc gcgacggcgg
     1501 ctactacagc ttcgtggtgg acagccacat gcacttcaag agcgccatcc accccagcat
     1561 cctgcagaac gggggcccca tgttcgcctt ccgccgcgtg gaggagctgc acagcaacac
     1621 cgagctgggc atcgtggagt accagcacgc cttcaagacc cccatcgcct tcgccagatc
     1681 tcgagctcga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     1741 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     1801 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt
     1861 aaagcaagta aaacctctac aaatgtggta cttaagaggg ggagaccaaa gggcgagacg
     1921 ttaaggaaag ctagctaatg ccaactttgt acaagaaagc tgttaattaa tcgcgactag
     1981 tcaggaaaca gctatgacgg tctagaaaaa aagcaccgac tcggtgccac tttttcaagt
     2041 tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaacag gtcttctcga
     2101 agacccggtg tttcgtcctt tccacaagat atataaagcc aagaaatcga aatactttca
     2161 agttacggta agcatatgat agtccatttt aaaacataat tttaaaactg caaactaccc
     2221 aagaaattat tactttctac gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa
     2281 attaattcta attatctctc taacagcctt gtatcgtata tgcaaatatg aaggaatcat
     2341 gggaaatagg ccctcactgg ccgtcgtttt actgatcaga tatcatgtga gcaaaaggcc
     2401 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     2461 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2521 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2581 tgccgcttac gggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2641 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2701 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2761 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     2821 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     2881 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     2941 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3001 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3061 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgccg tctcagaaga
     3121 actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa
     3181 gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca
     3241 acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa
     3301 agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat
     3361 cctcgccgtc gggcatgctc gccttgagcc tggcgaacag ttcggctggc gcgagcccct
     3421 gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc
     3481 gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca
     3541 gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca
     3601 ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa
     3661 cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct
     3721 cgtcttgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc
     3781 cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt
     3841 catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt
     3901 caatcataat attattgaag catttatcag ggtt
//
