LOCUS       40924_25181        3515 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses the S. pyogenes sgRNA from the human U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3515)
  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 3515)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 3515)
  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..3515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        696..936
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        963..1038
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1094..1112)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1133..1149)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(1157..1173)
                     /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(1181..1211)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(1226..1247)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(1364..1381)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1535..2123)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2203..2994)
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(3029..3386)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        complement(3388..3492)
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtacccg
      661 tctcacaggc ggatcgatcc aaggtcgggc aggaagaggg cctatttccc atgattcctt
      721 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
      781 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      841 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      901 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgggtct tcgagaagac
      961 ctgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     1021 gtggcaccga gtcggtgctt tttttctccg ctgagcgtac tgagacgccg cggtggagct
     1081 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc
     1141 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     1201 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     1261 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     1321 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     1381 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     1441 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     1501 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     1561 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     1621 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     1681 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     1741 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     1801 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     1861 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     1921 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     1981 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     2041 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     2101 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     2161 agtggaacga aaactcacgt taagggattt tggtcatgat cagaagaact cgtcaagaag
     2221 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
     2281 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
     2341 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
     2401 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
     2461 catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
     2521 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     2581 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     2641 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     2701 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     2761 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc
     2821 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     2881 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     2941 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     3001 cgatcctcat cctgtctctt gatcgatctt tgcaaaagcc taggcctcca aaaaagcctc
     3061 ctcactactt ctggaatagc tcagaggccg aggcggcctc ggcctctgca taaataaaaa
     3121 aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg
     3181 gcggagttag gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc
     3241 tgcctgctgg ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc
     3301 tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat
     3361 tccacagctg gttctttccg cctcaggact cttccttttt caatattatt gaagcattta
     3421 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     3481 aggggttccg cgcacatttc cccgaaaagt gccac
//
