LOCUS       sequence-484423-        3192 bp    DNA     circular SYN 22-AUG-2025
DEFINITION  Contains the U6 promoter and an optimized gRNA backbone inserted 
            with two copies of the MS2 stem loop..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3192)
  AUTHORS   Ling S, Zhang X, Dai Y, Jiang Z, Zhou X, Lu S, Qian X, Liu J, 
            Selfjord N, Satir TM, Lundin A, Touza JL, Firth M, Van Zuydam N, 
            Bilican B, Akcakaya P, Hong J, Cai Y
  TITLE     Customizable virus-like particles deliver CRISPR-Cas9 
            ribonucleoprotein for effective ocular neovascular and Huntington's 
            disease gene therapy.
  JOURNAL   Nat Nanotechnol. 2025 Apr;20(4):543-553. doi: 
            10.1038/s41565-024-01851-7. Epub 2025 Feb 10.
  PUBMED    39930103
REFERENCE   2  (bases 1 to 3192)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 22, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41565-024-01851-7"; journalName: "Nat Nanotechnol"; date: 
            "2025-04"; volume: "20"; issue: "4"; pages: "543-553"
COMMENT     Sequence Label: pU6-Osp.gRNA-MS2in
FEATURES             Location/Qualifiers
     source          1..3192
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        402..650
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     402..422
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     573..592
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        708..726
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        792..810
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     primer_bind     complement(971..987)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(971..987)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(984..1006)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    995..1011
                     /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(1019..1025,1026..1043,1044..1049))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1064..1085
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1202..1219)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1373..1961)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1453..1472)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2132..2923,2924..2992))
                     /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     2755..2774
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2993..3097)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3165..3183
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagaccgtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagggccta tttcccatga
      421 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
      481 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
      541 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
      601 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc ggagacggac
      661 atacaagtcg acaataggga taccgtctct gtttcagagc taggccaaca tgaggatcac
      721 ccatgtctgc agggcctgct ggaaacagca tagcaagttg aaataaggct agtccgttat
      781 caacttggcc aacatgagga tcacccatgt ctgcagggcc gaaaaagtgg caccgagtcg
      841 gtgctttttt tccgcgggga tccagacaca attgcattca ttttatgttt caggttcagg
      901 gggaggtgtg ggaggttttt tcggatcctc tagagtcgac ctgcaggcat gcaagcttgg
      961 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     1021 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     1081 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     1141 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     1201 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     1261 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     1321 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     1381 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     1441 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     1501 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     1561 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     1621 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     1681 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     1741 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     1801 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     1861 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     1921 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     1981 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     2041 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     2101 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     2161 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     2221 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     2281 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     2341 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     2401 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     2461 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     2521 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     2581 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     2641 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     2701 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     2761 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     2821 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     2881 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     2941 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     3001 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     3061 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     3121 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     3181 ggccctttag tc
//
