LOCUS       Exported                3140 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  sgRNA expression.
ACCESSION   .
VERSION     .
KEYWORDS    pRG2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3140)
  AUTHORS   Kim S, Bae T, Hwang J, Kim JS
  TITLE     Rescue of high-specificity Cas9 variants using sgRNAs with matched 
            5' nucleotides.
  JOURNAL   Genome Biol. 2017 Nov 15;18(1):218. doi: 10.1186/s13059-017-1355-3.
  PUBMED    29141659
REFERENCE   2  (bases 1 to 3140)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3140
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        66..306
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     66..86
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     237..256
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     388..406
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(444..466)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(561..878)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNRLA
                     AHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDA
                     A"
     primer_bind     566..585
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     779..801
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     793..810
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     794..810
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    814..870
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(879..895)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(879..895)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(892..914)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    903..919
                     /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(927..957)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     misc_RNA        987..1062
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1196..1213)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1367..1955)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1447..1466)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2126..2986)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2749..2768
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2987..3091)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gattagtgaa cggatctcga cggtatcgat cacgagacta gcctcgagcg gccgccccct
       61 tcaccgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
      121 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
      181 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
      241 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
      301 aaggacgaaa caccggagac cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
      361 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
      421 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
      481 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
      541 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
      601 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccattcg
      661 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
      721 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
      781 cagtcacgac gttgtaaaac gacggccagt gccaagcttg catgcctgca ggtcgactct
      841 agaggatccc cgggtaccga gctcgaattc gtaatcatgt catagctgtt tcctgtgtga
      901 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
      961 tggggtgcct aatagatccg gtctccgttt tagagctaga aatagcaagt taaaataagg
     1021 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttct agattcgcga
     1081 tgtacgggcc agatatacgc gttgacattg attattgact agttgtcttc ctgcattaat
     1141 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     1201 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     1261 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     1321 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     1381 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     1441 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     1501 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     1561 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     1621 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     1681 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     1741 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     1801 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     1861 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     1921 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     1981 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     2041 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     2101 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     2161 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     2221 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagat ccacgctcac
     2281 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     2341 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     2401 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     2461 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     2521 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     2581 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     2641 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     2701 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     2761 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     2821 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     2881 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     2941 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     3001 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     3061 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     3121 tcgacgaaga ctcaattgtc
//
