LOCUS       sequence-287407-        3973 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses a guide RNA (gRNA) to target human AAVS1 (T2 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 Apr 14, 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-T2
FEATURES             Location/Qualifiers
     source          1..3973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    342..366
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_RNA        complement(378..453)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(474..722)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(532..551)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(702..722)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(861..880)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(862..880)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(887..904)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(887..903)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(896..918)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1041..1343
                     /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     1222..1241
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1692..2486
                     /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             1692..2486
                     /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(1746..1765)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2356..2375
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2686..2695
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2692..3066
                     /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      3204..3792
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3693..3712
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3946..3963
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      301 ctagtaacgg ccgccagtgt gctggaattc gccctttaat gccaactttg tacaagaaag
      361 ctgggtctag aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc
      421 cttattttaa cttgctattt ctagctctaa aacatcctgt ccctagtggc cccggtgttt
      481 cgtcctttcc acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc
      541 atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac
      601 tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt aattctaatt
      661 atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc
      721 tcttcctgcc cgaccttggt accggatcca gtcgactgaa ttggttcctt taaagcctgc
      781 ttttttgtac aaagggcgaa ttctgcagat atccatcaca ctggcggccg ctcgagcatg
      841 catctagagg gcccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt
      901 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
      961 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     1021 tgcgcagcct atacgtacgg cagtttaagg tttacaccta taaaagagag agccgttatc
     1081 gtctgtttgt ggatgtacag agtgatatta ttgacacgcc ggggcgacgg atggtgatcc
     1141 ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc
     1201 atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtctccg
     1261 ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta
     1321 acctgatgtt ctggggaata taaatgtcag gcatgagatt atcaaaaagg atcttcacct
     1381 agatcctttt cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg atgaatgtca
     1441 gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca
     1501 gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat
     1561 tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt
     1621 tcttgccgcc aaggatctga tggcgcaggg gatcaagctc tgatcaagag acaggatgag
     1681 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     1741 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     1801 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     1861 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     1921 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     1981 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     2041 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     2101 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     2161 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga
     2221 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     2281 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     2341 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     2401 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     2461 atcgccttct tgacgagttc ttctgaatta ttaacgctta caatttcctg atgcggtatt
     2521 ttctccttac gcatctgtgc ggtatttcac accgcatcag gtggcacttt tcggggaaat
     2581 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2641 agacaataac cctgataaat gcttcaataa tagcacgtga ggagggccac catggccaag
     2701 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg
     2761 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg
     2821 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg
     2881 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc
     2941 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg
     3001 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag
     3061 gactgacacg tgctaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     3121 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3181 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3241 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3301 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     3361 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3421 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3481 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3541 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3601 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3661 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3721 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3781 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3841 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3901 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3961 gaggaagcgg aag
//
