LOCUS       sequence-222985-        3021 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single short guide RNA targeting CGTGATGTTGTACCGCTTC in iRFP670 
            sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3021)
  AUTHORS   Rui Y, Varanasi M, Mendes S, Yamagata HM, Wilson DR, Green JJ
  TITLE     Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of 
            CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion.
  JOURNAL   Mol Ther Nucleic Acids. 2020 Apr 21;20:661-672. doi: 
            10.1016/j.omtn.2020.04.005.
  PUBMED    32380416
REFERENCE   2  (bases 1 to 3021)
  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: "Mol Ther 
            Nucleic Acids."; date: "2020-04-21"; volume: "20:661-672. doi"; 
            pages: " 10.1016/j.omtn.2020.04.005"
COMMENT     Sequence Label: sgiRFP3
FEATURES             Location/Qualifiers
     source          1..3021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(50..66)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(50..66)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(63..85)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    74..90
                     /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(98..104,105..122,123..128))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    143..164
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        223..418
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      269..404
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    424..558
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(461..480)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     515..534
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(626..643)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(797..1385)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(877..896)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1556..2347,2348..2416))
                     /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     2179..2198
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2417..2521)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2653..2901
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2653..2673
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2824..2843
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2922..2997
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(join(3009..3021,1..12))
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 gtacaaagtt ggcattatgc ccccgggcgc caagcttggc gtaatcatgg tcatagctgt
       61 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
      121 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
      181 tgcccgcttt ccagtcggga aacctgtcgt gccagcggat ccgcatctca attagtcagc
      241 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
      301 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
      361 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
      421 gctaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
      481 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
      541 tcttatcatg tctggatccg ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
      601 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
      661 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
      721 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
      781 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
      841 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
      901 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
      961 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     1021 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     1081 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     1141 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     1201 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     1261 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     1321 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     1381 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     1441 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     1501 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     1561 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     1621 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     1681 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     1741 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     1801 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     1861 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     1921 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     1981 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     2041 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     2101 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     2161 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     2221 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     2281 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     2341 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     2401 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     2461 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     2521 gcacatttcc ccgaaaagtg ccacctgggt cgacattgat tattgactag tagtggagaa
     2581 ttctgtacaa aaaagcaggc tttaaaggaa ccaattcagt cgactggatc cggtaccaag
     2641 gtcgggcagg aagagggcct atttcccatg attccttcat atttgcatat acgatacaag
     2701 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat
     2761 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     2821 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     2881 ttgtggaaag gacgaaacac cggaagcggt acaacatcac ggttttagag ctagaaatag
     2941 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     3001 ttttctagac ccagctttct t
//
