LOCUS       sequence-222984-        3021 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single short guide RNA targeting GCGCGTTCTTTGGACGCGA 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: sgiRFP2
FEATURES             Location/Qualifiers
     source          1..3021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        65..313
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     65..85
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     236..255
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        334..409
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(421..445)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(483..499)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(483..499)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(496..518)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    507..523
                     /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(531..537,538..555,556..561))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    576..597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        656..851
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      702..837
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    857..991
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(894..913)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     948..967
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1059..1076)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1230..1818)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1310..1329)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1989..2780,2781..2849))
                     /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     2612..2631
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2850..2954)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaaaagcag gctttaaagg aaccaattca gtcgactgga tccggtacca aggtcgggca
       61 ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
      121 agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
      181 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
      241 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
      301 aggacgaaac accgtcgcgt ccaaagaacg cgcgttttag agctagaaat agcaagttaa
      361 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttctag
      421 acccagcttt cttgtacaaa gttggcatta tgcccccggg cgccaagctt ggcgtaatca
      481 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
      541 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
      601 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc
      661 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
      721 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
      781 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
      841 gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat aaagcaatag
      901 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
      961 actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg
     1021 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     1081 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     1141 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     1201 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     1261 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     1321 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     1381 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     1441 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     1501 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     1561 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     1621 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     1681 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     1741 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     1801 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     1861 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     1921 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     1981 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2041 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     2101 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     2161 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     2221 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     2281 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     2341 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     2401 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     2461 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     2521 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     2581 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     2641 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     2701 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     2761 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     2821 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     2881 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     2941 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ggtcgacatt gattattgac
     3001 tagtagtgga gaattctgta c
//
