LOCUS       40924_24548        3451 bp DNA     circular SYN 12-JUN-2021
DEFINITION  Expresses the S. pyogenes sgRNA from the H1 promoter .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3451)
  AUTHORS   Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA
  TITLE     Multiplex CRISPR/Cas9-based genome engineering from a single 
            lentiviral vector.
  JOURNAL   Nucleic Acids Res. 2014 Aug 13. pii: gku749.
  PUBMED    25122746
REFERENCE   2  (bases 1 to 3451)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 3451)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2014 Aug 13. pii: gku749."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..215
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     misc_RNA        242..317
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(362..380)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(401..417)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(425..441)
                     /label=lac operator
                     /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(449..479)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(494..515)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(632..649)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(803..1391)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1471..2262)
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(2297..2654)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        complement(2656..2760)
                     /label=AmpR promoter
     rep_origin      complement(2786..3241)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     3383..3399
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3409..3427
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gaacgctgac gtcatcaacc cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa
       61 cacccagcgc gcgtgcgccc tggcaggaag atggctgtga gggacagggg agtggcgccc
      121 tgcaatattt gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatgtctttg
      181 gatttgggaa tcttataagt tctgtatgag accactcttt cccaggtctt cgagaagacc
      241 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
      301 tggcaccgag tcggtgcttt ttttggtctg agacgccgcg gtggagctcc agcttttgtt
      361 ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt
      421 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
      481 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
      541 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
      601 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
      661 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
      721 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
      781 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
      841 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
      901 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
      961 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     1021 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     1081 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     1141 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     1201 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     1261 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     1321 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     1381 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     1441 actcacgtta agggattttg gtcatgatca gaagaactcg tcaagaaggc gatagaaggc
     1501 gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc
     1561 gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc
     1621 cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt
     1681 cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgctcgcctt
     1741 gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg
     1801 atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg
     1861 gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat
     1921 ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc
     1981 caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac
     2041 gcccgtcgtg gccagccacg atagccgcgc tgcctcgtct tgcagttcat tcagggcacc
     2101 ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc
     2161 ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca
     2221 agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc
     2281 tgtctcttga tcgatctttg caaaagccta ggcctccaaa aaagcctcct cactacttct
     2341 ggaatagctc agaggccgag gcggcctcgg cctctgcata aataaaaaaa attagtcagc
     2401 catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg
     2461 gcgggactat ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg
     2521 agcctgggga ctttccacac ctggttgctg actaattgag atgcatgctt tgcatacttc
     2581 tgcctgctgg ggagcctggg gactttccac accctaactg acacacattc cacagctggt
     2641 tctttccgcc tcaggactct tcctttttca atattattga agcatttatc agggttattg
     2701 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     2761 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg
     2821 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     2881 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     2941 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     3001 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     3061 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     3121 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     3181 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     3241 tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     3301 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
     3361 gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca
     3421 ctatagggcg aattgggtac ccgtctcacc t
//
