LOCUS       sequence-273631-        3538 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a non-targeting sgRNA to the left and right of iCas9 
            recognition site to be used as a control in Traffic Light reporter 
            system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3538)
  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 3538)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 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(NT:NT)
FEATURES             Location/Qualifiers
     source          1..3538
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     94..113
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     307..329
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     321..338
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     322..338
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    339..395
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     promoter        396..644
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        663..738
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        819..1067
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1086..1161
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1260..1276)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1260..1276)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1273..1295)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1284..1300
                     /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(1308..1314,1315..1332,1333..1338))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1353..1374
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1491..1508)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1662..2250)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1742..1761)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2421..3212,3213..3281))
                     /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     3044..3063
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3282..3386)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3454..3472
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3510..3532)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
       61 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
      121 tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
      181 gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
      241 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
      301 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attcgagctc ggtacccggg
      361 gatcctctag agtcgacctg caggcatgca agcttgaggg cctatttccc atgattcctt
      421 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
      481 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      541 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      601 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgggtct tcgagaagac
      661 ctgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
      721 gtggcaccga gtcggtgctt ttttgtttta gagctagaaa tagcaagtta aaataaggct
      781 agtccgtttt tagcgcgtgc gccaattctg cagacagaga gggcctattt cccatgattc
      841 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
      901 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
      961 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     1021 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg tcttcgagaa
     1081 gacctgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     1141 aaagtggcac cgagtcggtg cttttttgtt ttagagctag aaatagcaag ttaaaataag
     1201 gctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa aaagcttggc gtaatcatgg
     1261 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     1321 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     1381 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     1441 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     1501 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     1561 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     1621 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     1681 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     1741 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     1801 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     1861 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     1921 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     1981 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     2041 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     2101 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     2161 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     2221 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     2281 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     2341 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     2401 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     2461 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     2521 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     2581 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     2641 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     2701 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     2761 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     2821 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     2881 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     2941 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     3001 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     3061 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     3121 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     3181 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     3241 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     3301 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     3361 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     3421 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     3481 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacgg
//
