LOCUS       sequence-454893-        3390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP dropout backbone for sgRNA library cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3390)
  AUTHORS   Nugent PJ, Park H, Wladyka CL, Yelland JN, Sinha S, Chen KY, Bynum 
            C, Quarterman G, Lee SC, Hsieh AC, Subramaniam AR
  TITLE     Decoding post-transcriptional regulatory networks by RNA-linked 
            CRISPR screening in human cells.
  JOURNAL   Nat Methods. 2025 Jun;22(6):1237-1246. doi: 
            10.1038/s41592-025-02702-6. Epub 2025 May 29.
  PUBMED    40442371
REFERENCE   2  (bases 1 to 3390)
  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"; doi: 
            "10.1038/s41592-025-02702-6"; journalName: "Nat Methods"; date: 
            "2025-06"; volume: "22"; issue: "6"; pages: "1237-1246"
COMMENT     Sequence Label: pHPHS309
FEATURES             Location/Qualifiers
     source          1..3390
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      77..665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     566..585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        768..1016
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     768..788
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     939..958
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     RBS             1171..1182
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1189..1902
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1914..1985
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2001..2028
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        2040..2115
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2192..2210)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2288..2307)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2342..2361
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2427..3242)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2427..3242)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3150..3169
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3234..3253
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
       61 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      721 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtctagag ggcctatttc
      781 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attagaatta
      841 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
      901 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
      961 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggat
     1021 ccgaaagtga aacgtgattt catgcgtcat tttgaacatt ttgtaaatct tatttaataa
     1081 tgtgtgcggc aattcacatt taatttatga atgttttctt aacatcgcgg caactcaaga
     1141 aacggcaggt tcggatctta gctactagag aaagaggaga aatactagat gcgtaaaggc
     1201 gaagagctgt tcactggtgt cgtccctatt ctggtggaac tggatggtga tgtcaacggt
     1261 cataagtttt ccgtgcgtgg cgagggtgaa ggtgacgcaa ctaatggtaa actgacgctg
     1321 aagttcatct gtactactgg taaactgccg gttccttggc cgactctggt aacgacgctg
     1381 acttatggtg ttcagtgctt tgctcgttat ccggaccata tgaagcagca tgacttcttc
     1441 aagtccgcca tgccggaagg ctatgtgcag gaacgcacga tttcctttaa ggatgacggc
     1501 acgtacaaaa cgcgtgcgga agtgaaattt gaaggcgata ccctggtaaa ccgcattgag
     1561 ctgaaaggca ttgactttaa agaggacggc aatatcctgg gccataagct ggaatacaat
     1621 tttaacagcc acaatgttta catcaccgcc gataaacaaa aaaatggcat taaagcgaat
     1681 tttaaaattc gccacaacgt ggaggatggc agcgtgcagc tggctgatca ctaccagcaa
     1741 aacactccaa tcggtgatgg tcctgttctg ctgccagaca atcactatct gagcacgcaa
     1801 agcgttctgt ctaaagatcc gaacgagaaa cgcgatcata tggttctgct ggagttcgta
     1861 accgcagcgg gcatcacgca tggtatggat gaactgtaca aatgaccagg catcaaataa
     1921 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     1981 ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatactcgag
     2041 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     2101 gcaccgagtc ggtgcttttt tgcgagctca cggggacagc ggccgcggaa agatcgccgt
     2161 gtaagcgata tcagatcgga agagcgtcgt gcctatagtg agtcgtatta gcctagttgc
     2221 cagccatctg gcgcgccatc agagagatcc tcgcgaattc ggcgatccag acatgataag
     2281 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     2341 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacggtaccg
     2401 tgttacaacc aattaaccaa ttctgattag aaaaactcat cgagcatcaa atgaaactgc
     2461 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     2521 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     2581 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     2641 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag cttatgcatt
     2701 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     2761 accaaaccgt tattcattcg tgattgcgcc tgagcgaggc gaaatacgcg atcgctgtta
     2821 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     2881 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     2941 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     3001 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     3061 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     3121 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     3181 gcatccatgt tggaatttaa tcgcggcctg gagcaagacg tttcccgttg aatatggctc
     3241 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgatgatata
     3301 tttttatctt gtgcaatgta acatcagaga ttttgagaca caacgtggct ttgttgaata
     3361 aatcgaactt ttgctgagtt gaaggatcag
//
