LOCUS       sequence-201301-        4545 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3A igRNA and Hybrid-promoter sfGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4545)
  AUTHORS   Galizi R, Duncan JN, Rostain W, Quinn CM, Storch M, Kushwaha M, 
            Jaramillo A
  TITLE     Engineered RNA-Interacting CRISPR Guide RNAs for Genetic Sensing and
            Diagnostics.
  JOURNAL   CRISPR J. 2020 Oct;3(5):398-408. doi: 10.1089/crispr.2020.0029.
  PUBMED    33095053
REFERENCE   2  (bases 1 to 4545)
  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: 
            "10.1089/crispr.2020"; journalName: "CRISPR J"; date: "2020-10"; 
            volume: "3"; issue: "5"; pages: "398-408"
COMMENT     Sequence Label: PAJ469
FEATURES             Location/Qualifiers
     source          1..4545
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             428..439
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        631..665
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        729..804
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           809..876
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     terminator      1051..1145
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      1637..1859
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             1907..2857
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFGIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     primer_bind     complement(3204..3223)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3215..4030
                     /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"
     primer_bind     complement(3288..3307)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(4129..4160)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(join(4247..4545,1..412))
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="RKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
ORIGIN
        1 ggcccaggat attgccgtct tctttaaagt caatgccttt cagctcaatg cggtttacca
       61 gggtatcgcc ttcaaatttc acttccgcac gcgttttgta cgtgccgtca tccttaaagg
      121 aaatcgtgcg ttcctgcaca tagccttccg gcatggcgga cttgaagaag tcatgctgct
      181 tcatatggtc cggataacga gcaaagcact gaacaccata agtcagcgtc gttaccagag
      241 tcggccaagg taccggcagt ttaccagtag tacagatgaa cttcagcgtc agtttaccat
      301 tagttgcgtc accttcaccc tcgccacgca cggaaaactt atgaccgttg acatcaccat
      361 ccagttccac cagaataggg acgacaccag tgaacagctc ttcgccttta cgactagtca
      421 tctagtattt ctcctctttc tctagtaggt cagtgcgtcc tgctgatgtg ctcagtatcc
      481 ataccgcgaa aggttttgca caaatcctag atcagacttt gacgccaggc aagtctcagc
      541 tgatgtgctc agtatccata ccgcgaaagg ttttgcacaa atcctagatc gtgcagactt
      601 tgacgccagg caagtctcag ctgggtcgac ttgacagcta gctcagtcct aggtataatg
      661 ctagcgcgac ttcatgctgt aaaggttttg cacgtttctt tgtctgccgt gcaaaacctt
      721 tcgcggtagt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
      781 gaaaaagtgg caccgagtcg gtgcttttgg ccggcatggt cccagcctcc tcgctggcgc
      841 cggctgggca acatgcttcg gcatggcgaa tgggacttgt tcagaacgct cggtcttgca
      901 caccgggcgt tttttctttg tgagtccaga cgtcaggtgg caaacgcgaa gtaatctttt
      961 cggttttaaa gaaaaagggc agggtggtga caccttgccc gtttttttgc cggagctaga
     1021 gctgtcagac caagtttacg agctcgcttg gactcctgtt gatagatcca gtaatgacct
     1081 cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg
     1141 agaatccaag cactagggac agtaagacgg gtaagcctgt tgatgatacc gctgccttac
     1201 tgggtgcatt agccagtctg aatgacctgt cacgggataa tccgaagtgg tcagactgga
     1261 aaatcagagg gcaggaactg ctgaacagca aaaagtcaga tagcaccaca tagcagaccc
     1321 gccataaaac gccctgagaa gcccgtgacg ggcttttctt gtattatggg tagtttcctt
     1381 gcatgaatcc ataaaaggcg cctgtagtgc catttacccc cattcactgc cagagccgtg
     1441 agcgcagcga actgaatgtc acgaaaaaga cagcgactca ggtgcctgat ggtcggagac
     1501 aaaaggaata ttcagcgatt tgcccgagct tgcgagggtg ctacttaagc ctttagggtt
     1561 ttaaggtctg ttttgtagag gagcaaacag cgtttgcgac atccttttgt aatactgcgg
     1621 aactgactaa agtagtgagt tatacacagg gctgggatct attcttttta tcttttttta
     1681 ttctttcttt attctataaa ttataaccac ttgaatataa acaaaaaaaa cacacaaagg
     1741 tctagcggaa tttacagagg gtctagcaga atttacaagt tttccagcaa aggtctagca
     1801 gaatttacag atacccacaa ctcaaaggaa aaggacatgt aattatcatt gactagccca
     1861 tctcaattgg tatagtgatt aaaatcacct agaccaattg agatgtatgt ctgaattagt
     1921 tgttttcaaa gcaaatgaac tagcgattag tcgctatgac ttaacggagc atgaaaccaa
     1981 gctaatttta tgctgtgtgg cactactcaa ccccacgatt gaaaacccta caaggaaaga
     2041 acggacggta tcgttcactt ataaccaata cgctcagatg atgaacatca gtagggaaaa
     2101 tgcttatggt gtattagcta aagcaaccag agagctgatg acgagaactg tggaaatcag
     2161 gaatcctttg gttaaaggct ttgggatttt ccagtggaca aactatgcca agttctcaag
     2221 cgaaaaatta gaattagttt ttagtgaaga gatattgcct tatcttttcc agttaaaaaa
     2281 attcataaaa tataatctgg aacatgttaa gtcttttgaa aacaaatact ctatgaggat
     2341 ttatgagtgg ttattaaaag aactaacaca aaagaaaact cacaaggcaa atatagagat
     2401 tagccttgat gaatttaagt tcatgttaat gcttgaaaat aactaccatg agtttaaaag
     2461 gcttaaccaa tgggttttga aaccaataag taaagattta aacacttaca gcaatatgaa
     2521 attggtggtt gataagcgag gccgcccgac tgatacgttg attttccaag ttgaactaga
     2581 tagacaaatg gatctcgtaa ccgaacttga gaacaaccag ataaaaatga atggtgacaa
     2641 aataccaaca accattacat cagattccta cctacataac ggactaagaa aaacactaca
     2701 cgatgcttta actgcaaaaa ttcagctcac cagttttgag gcaaaatttt tgagtgacat
     2761 gcaaagtaag tatgatctca atggttcgtt ctcatggctc acgcaaaaac aacgaaccac
     2821 actagagaac atactggcta aatacggaag gatctgaggt tcttatggct cttgtatcta
     2881 tcagtgaagc atcaagacta acaaacaaaa gtagaacaac tgttcaccgt tacatatcaa
     2941 agggaaaact gtccatatgc acagatgaaa acggtgtaaa aaagatagat acatcagagc
     3001 ttttacgagt ttttggtgca ttcaaagctg ttcaccatga acagatcgac aatgtaacta
     3061 ctagagctga tccttcaact cagcaaaagt tcgatttatt caacaaagcc acgttgtgtc
     3121 tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact
     3181 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
     3241 ttgctcccgt ccgcgcttaa actccaacat ggacgctgat ttatatgggt ataaatgggc
     3301 tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc
     3361 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     3421 ggtccgtctc aactggctga cggagtttat gcctctcccg accatcaagc attttatccg
     3481 tactcctgat gatgcgtggt tactcaccac cgcgattcct gggaaaacag ccttccaggt
     3541 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggccg tgttcctgcg
     3601 ccggttacat tcgattcctg tttgtaattg tccttttaac agcgatcgtg tatttcgtct
     3661 tgctcaggcg caatcacgca tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     3721 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cacaagctct tgccattctc
     3781 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     3841 gaaattaata ggttgtattg atgttggacg ggtcggaatc gcagaccgtt accaggacct
     3901 tgccattctt tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     3961 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     4021 gtttttctaa taatactagc agaaatcatc cttagcgaaa gctaaggatt ttttttatct
     4081 gattaccgcc tttgagtgag ccccaatgat aaccccaagg gaagttttag tcaaaagcct
     4141 ccggtcggag gcttttgact ttctgctaat ctaactaact aacccttagt gactcctgca
     4201 gttattacgc tgcaagggcg taattttcgt cgttcgctgc gctagctttg tacagttcat
     4261 ccataccatg cgtgatgccc gctgcggtta cgaactccag cagaaccata tgatcgcgtt
     4321 tctcgttcgg atctttagac agaacgcttt gcgtgctcag atagtgattg tctggcagca
     4381 gaacaggacc atcaccgatt ggagtgtttt gctggtagtg atcagccagc tgcacgctgc
     4441 catcctccac gttgtggcga attttaaaat tcgctttaat gccatttttt tgtttatcgg
     4501 cggtgatgta aacattgtgg ctgttaaaat tgtattccag cttat
//
