LOCUS       sequence-224317-        3973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a guide RNA (gRNA) to target human AAVS1 (T1 target 
            sequence) for genome engineering.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3973)
  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 3973)
  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_AAVS1-T1
FEATURES             Location/Qualifiers
     source          1..3973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(357..381)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(466..484)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(467..484)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(502..518)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(502..518)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(515..537)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    526..542
                     /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(550..556,557..574,575..580))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    595..616
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(733..750)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(904..1492)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(984..1003)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1630..2004)
                     /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"
     regulatory      2001..2010
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2210..3004)
                     /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(2210..3004)
                     /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(2321..2340)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2931..2950
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3353..3655)
                     /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     complement(3455..3474)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3778..3800
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3792..3809
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3793..3809
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3816..3835
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3816..3834
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg tcccctccac cccacagtgg ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctagaccc
      361 agctttcttg tacaaagttg gcattaaagg gcgaattcca gcacactggc ggccgttact
      421 agtggatccg agctcggtac caagcttgat gcatagcttg agtattctat agtgtcacct
      481 aaatagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
      541 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
      601 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
      661 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
      721 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
      781 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
      841 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      901 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      961 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     1021 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     1081 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     1141 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     1201 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     1261 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     1321 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     1381 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     1441 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     1501 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     1561 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     1621 tagcacgtgt cagtcctgct cctcggccac gaagtgcacg cagttgccgg ccgggtcgcg
     1681 cagggcgaac tcccgccccc acggctgctc gccgatctcg gtcatggccg gcccggaggc
     1741 gtcccggaag ttcgtggaca cgacctccga ccactcggcg tacagctcgt ccaggccgcg
     1801 cacccacacc caggccaggg tgttgtccgg caccacctgg tcctggaccg cgctgatgaa
     1861 cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc tccacgaagt cccgggagaa
     1921 cccgagccgg tcggtccaga actcgaccgc tccggcgacg tcgcgcgcgg tgagcaccgg
     1981 aacggcactg gtcaacttgg ccatggtggc cctcctcacg tgctattatt gaagcattta
     2041 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     2101 aggggttccg cgcacatttc cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca
     2161 gatgcgtaag gagaaaatac cgcatcagga aattgtaagc gttaataatt cagaagaact
     2221 cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca
     2281 cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg
     2341 ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc
     2401 ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct
     2461 cgccgtcggg catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat
     2521 gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct
     2581 cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc
     2641 gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga
     2701 gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt
     2761 cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt
     2821 cttgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct
     2881 gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat
     2941 agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa
     3001 tcatgcgaaa cgatcctcat cctgtctctt gatcagagct tgatcccctg cgccatcaga
     3061 tccttggcgg caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg
     3121 gcgccccagc tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc
     3181 gccatgtaag cccactgcaa gctacctgct ttctctttgc gcttgcgttt tcccttgtcc
     3241 agatagccca gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct
     3301 acgtgaaaag gatctaggtg aagatccttt ttgataatct catgcctgac atttatattc
     3361 cccagaacat caggttaatg gcgtttttga tgtcattttc gcggtggctg agatcagcca
     3421 cttcttcccc gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc
     3481 tttcatcccc gatatgcacc accgggtaaa gttcacggga gactttatct gacagcagac
     3541 gtgcactggc cagggggatc accatccgtc gccccggcgt gtcaataata tcactctgta
     3601 catccacaaa cagacgataa cggctctctc ttttataggt gtaaacctta aactgccgta
     3661 cgtataggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     3721 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     3781 agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat
     3841 tgggccctct agatgcatgc tcgagcggcc gccagtgtga tggatatctg cagaattcgc
     3901 cctttgtaca aaaaagcagg ctttaaagga accaattcag tcgactggat ccggtaccaa
     3961 ggtcgggcag gaa
//
