LOCUS       sequence-269723-        3914 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An empty gRNA expression vector, used to create a gRNA to a specific
            sequence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3914)
  AUTHORS   Mali P, Yang L, Esvelt KM, Aach J, Guell M, Dicarlo JE, Norville JE,
            Church GM
  TITLE     RNA-Guided Human Genome Engineering via Cas9.
  JOURNAL   Science. 2013 Feb 15;339(6121):823-6. doi: 10.1126/science.1232033
  PUBMED    23287722
REFERENCE   2  (bases 1 to 3914)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2013-02-15- 15"; volume: "339"; 
            issue: "6121"; pages: "823-6"
COMMENT     Sequence Label: gRNA_Cloning Vector
FEATURES             Location/Qualifiers
     source          1..3914
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     380..399
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     633..650
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    767..788
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(803..808,809..826,827..833)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    841..857
                     /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     846..868
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     865..881
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     865..881
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        899..917
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     899..916
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    1002..1026
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1133..1152)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1303..1323)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1462..1481)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1463..1481)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1488..1505)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1488..1504)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1497..1519)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1642..1944
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1823..1842
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2293..3087
                     /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             2293..3087
                     /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(2347..2366)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2957..2976
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3287..3296
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3293..3667
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      join(3805..3914,1..479)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
       61 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
      121 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
      181 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
      241 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
      301 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
      361 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
      421 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
      481 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
      541 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
      601 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
      661 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
      721 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      781 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      841 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      901 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      961 ctagtaacgg ccgccagtgt gctggaattc gccctttaat gccaactttg tacaagaaag
     1021 ctgggtctag aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc
     1081 cttattttaa cttaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag
     1141 catatgatag tccattttaa aacataattt taaaactgca aactacccaa gaaattatta
     1201 ctttctacgt cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattctaat
     1261 tatctctcta acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc
     1321 ctcttcctgc ccgaccttgg taccggatcc agtcgactga attggttcct ttaaagcctg
     1381 cttttttgta caaagggcga attctgcaga tatccatcac actggcggcc gctcgagcat
     1441 gcatctagag ggcccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt
     1501 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     1561 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     1621 ttgcgcagcc tatacgtacg gcagtttaag gtttacacct ataaaagaga gagccgttat
     1681 cgtctgtttg tggatgtaca gagtgatatt attgacacgc cggggcgacg gatggtgatc
     1741 cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg
     1801 catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc
     1861 gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt
     1921 aacctgatgt tctggggaat ataaatgtca ggcatgagat tatcaaaaag gatcttcacc
     1981 tagatccttt tcacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc
     2041 agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc
     2101 agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa
     2161 ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct
     2221 ttcttgccgc caaggatctg atggcgcagg ggatcaagct ctgatcaaga gacaggatga
     2281 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     2341 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     2401 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     2461 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     2521 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     2581 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     2641 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     2701 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     2761 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     2821 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     2881 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     2941 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     3001 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     3061 tatcgccttc ttgacgagtt cttctgaatt attaacgctt acaatttcct gatgcggtat
     3121 tttctcctta cgcatctgtg cggtatttca caccgcatca ggtggcactt ttcggggaaa
     3181 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3241 gagacaataa ccctgataaa tgcttcaata atagcacgtg aggagggcca ccatggccaa
     3301 gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg
     3361 gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg
     3421 ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct
     3481 ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc
     3541 cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg
     3601 gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca
     3661 ggactgacac gtgctaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     3721 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3781 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3841 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3901 tctttttccg aagg
//
