LOCUS       sequence-222999-        3021 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single short guide RNA targeting GATCGAGTTCGAGCCTGCGG 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: sgiRFP1
FEATURES             Location/Qualifiers
     source          1..3021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(8..32)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(70..86)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(70..86)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(83..105)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    94..110
                     /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(118..124,125..142,143..148))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    163..184
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        243..438
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      289..424
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    444..578
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(481..500)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     535..554
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(646..663)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(817..1405)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(897..916)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1576..2367,2368..2436))
                     /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     2199..2218
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2437..2541)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2673..2921
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2673..2693
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2844..2863
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2942..3017
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tttctagacc cagctttctt gtacaaagtt ggcattatgc ccccgggcgc caagcttggc
       61 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
      121 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
      181 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagcggat
      241 ccgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
      301 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
      361 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
      421 ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc ttataatggt tacaaataaa
      481 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
      541 tgtccaaact catcaatgta tcttatcatg tctggatccg ctgcattaat gaatcggcca
      601 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
      661 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
      721 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
      781 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
      841 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
      901 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
      961 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     1021 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     1081 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     1141 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     1201 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     1261 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     1321 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     1381 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     1441 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     1501 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     1561 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     1621 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     1681 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     1741 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     1801 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     1861 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     1921 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     1981 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     2041 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     2101 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     2161 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     2221 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     2281 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     2341 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     2401 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     2461 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     2521 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgggt cgacattgat
     2581 tattgactag tagtggagaa ttctgtacaa aaaagcaggc tttaaaggaa ccaattcagt
     2641 cgactggatc cggtaccaag gtcgggcagg aagagggcct atttcccatg attccttcat
     2701 atttgcatat acgatacaag gctgttagag agataattag aattaatttg actgtaaaca
     2761 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     2821 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     2881 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgatcgagtt cgagcctgcg
     2941 ggttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
     3001 tggcaccgag tcggtgcttt t
//
