LOCUS       sequence-273638-        3544 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of humanized mCherry-targeting sgRNAs to the left (G) and
            right (H) of iCas9 recognition sequences to be used in Traffic Light
            reporter system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3544)
  AUTHORS   Standage-Beier K, Brookhouser N, Balachandran P, Zhang Q, Brafman 
            DA, Wang X
  TITLE     RNA-Guided Recombinase-Cas9 Fusion Targets Genomic DNA Deletion and 
            Integration.
  JOURNAL   CRISPR J. 2019 Aug;2:209-222. doi: 10.1089/crispr.2019.0013.
  PUBMED    31436506
REFERENCE   2  (bases 1 to 3544)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "CRISPR J.";
            date: "2019-08"; volume: "2:209-222. doi"; pages: " 
            10.1089/crispr.2019.0013"
COMMENT     Sequence Label: pUC19-hsg(G:H)
FEATURES             Location/Qualifiers
     source          1..3544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        149..397
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        419..494
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(593..609)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(593..609)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(606..628)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    617..633
                     /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(641..647,648..665,666..671))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    686..707
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(824..841)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(995..1583)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1075..1094)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1754..2545,2546..2614))
                     /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     2377..2396
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2615..2719)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2787..2805
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2843..2865)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2965..2984
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3178..3200
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3192..3209
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3193..3209
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    3210..3266
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     promoter        3267..3515
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        join(3537..3544,1..68)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
       61 gtcggtgctt ttttgtttta gagctagaaa tagcaagtta aaataaggct agtccgtttt
      121 tagcgcgtgc gccaattctg cagacagaga gggcctattt cccatgattc cttcatattt
      181 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
      241 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
      301 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
      361 cttggcttta tatatcttgt ggaaaggacg aaacaccgac agaccttact ccagaagggt
      421 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      481 caccgagtcg gtgctttttt gttttagagc tagaaatagc aagttaaaat aaggctagtc
      541 cgtttttagc gcgtgcgcca attctgcaga caaaaagctt ggcgtaatca tggtcatagc
      601 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
      661 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
      721 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
      781 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
      841 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
      901 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
      961 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     1021 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     1081 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     1141 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     1201 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     1261 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     1321 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     1381 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     1441 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     1501 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     1561 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     1621 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     1681 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     1741 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     1801 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     1861 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     1921 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     1981 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     2041 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     2101 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     2161 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     2221 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     2281 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     2341 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     2401 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     2461 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     2521 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     2581 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     2641 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     2701 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     2761 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc
     2821 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     2881 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     2941 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     3001 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc
     3061 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     3121 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     3181 agtcacgacg ttgtaaaacg acggccagtg aattcgagct cggtacccgg ggatcctcta
     3241 gagtcgacct gcaggcatgc aagcttgagg gcctatttcc catgattcct tcatatttgc
     3301 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     3361 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     3421 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     3481 tggctttata tatcttgtgg aaaggacgaa acaccgacgt acgagtgcaa gcctgggttt
     3541 taga
//
