LOCUS       sequence-200486-        4467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3A_v9 igRNA and sfGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4467)
  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 4467)
  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: PAJ407
FEATURES             Location/Qualifiers
     source          1..4467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             445..456
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    497..513
                     /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        570..604
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        668..743
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           748..815
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     terminator      990..1084
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      1576..1798
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             1846..2796
                     /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(3143..3162)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3154..3969
                     /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(3227..3246)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(4068..4099)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(join(4186..4467,1..429))
                     /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 attgtattcc agcttatggc ccaggatatt gccgtcttct ttaaagtcaa tgcctttcag
       61 ctcaatgcgg tttaccaggg tatcgccttc aaatttcact tccgcacgcg ttttgtacgt
      121 gccgtcatcc ttaaaggaaa tcgtgcgttc ctgcacatag ccttccggca tggcggactt
      181 gaagaagtca tgctgcttca tatggtccgg ataacgagca aagcactgaa caccataagt
      241 cagcgtcgtt accagagtcg gccaaggtac cggcagttta ccagtagtac agatgaactt
      301 cagcgtcagt ttaccattag ttgcgtcacc ttcaccctcg ccacgcacgg aaaacttatg
      361 accgttgaca tcaccatcca gttccaccag aatagggacg acaccagtga acagctcttc
      421 gcctttacga ctagtcatct agtatttctc ctctttctct agtaggtcag tgcgtcctgc
      481 tgatgtgctc agtatcttgt tatccgctca caatgtcaat gttatccgct cacatttatt
      541 gacgccaggc aagtctcagc tgggtcgact tgacagctag ctcagtccta ggtataatgc
      601 tagcgatacg ttccagacga aaggttttgc acgtaataga cgccagggtg caaaaccttt
      661 cgcggtagtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
      721 aaaaagtggc accgagtcgg tgcttttggc cggcatggtc ccagcctcct cgctggcgcc
      781 ggctgggcaa catgcttcgg catggcgaat gggacttgtt cagaacgctc ggtcttgcac
      841 accgggcgtt ttttctttgt gagtccagac gtcaggtggc aaacgcgaag taatcttttc
      901 ggttttaaag aaaaagggca gggtggtgac accttgcccg tttttttgcc ggagctagag
      961 ctgtcagacc aagtttacga gctcgcttgg actcctgttg atagatccag taatgacctc
     1021 agaactccat ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga
     1081 gaatccaagc actagggaca gtaagacggg taagcctgtt gatgataccg ctgccttact
     1141 gggtgcatta gccagtctga atgacctgtc acgggataat ccgaagtggt cagactggaa
     1201 aatcagaggg caggaactgc tgaacagcaa aaagtcagat agcaccacat agcagacccg
     1261 ccataaaacg ccctgagaag cccgtgacgg gcttttcttg tattatgggt agtttccttg
     1321 catgaatcca taaaaggcgc ctgtagtgcc atttaccccc attcactgcc agagccgtga
     1381 gcgcagcgaa ctgaatgtca cgaaaaagac agcgactcag gtgcctgatg gtcggagaca
     1441 aaaggaatat tcagcgattt gcccgagctt gcgagggtgc tacttaagcc tttagggttt
     1501 taaggtctgt tttgtagagg agcaaacagc gtttgcgaca tccttttgta atactgcgga
     1561 actgactaaa gtagtgagtt atacacaggg ctgggatcta ttctttttat ctttttttat
     1621 tctttcttta ttctataaat tataaccact tgaatataaa caaaaaaaac acacaaaggt
     1681 ctagcggaat ttacagaggg tctagcagaa tttacaagtt ttccagcaaa ggtctagcag
     1741 aatttacaga tacccacaac tcaaaggaaa aggacatgta attatcattg actagcccat
     1801 ctcaattggt atagtgatta aaatcaccta gaccaattga gatgtatgtc tgaattagtt
     1861 gttttcaaag caaatgaact agcgattagt cgctatgact taacggagca tgaaaccaag
     1921 ctaattttat gctgtgtggc actactcaac cccacgattg aaaaccctac aaggaaagaa
     1981 cggacggtat cgttcactta taaccaatac gctcagatga tgaacatcag tagggaaaat
     2041 gcttatggtg tattagctaa agcaaccaga gagctgatga cgagaactgt ggaaatcagg
     2101 aatcctttgg ttaaaggctt tgggattttc cagtggacaa actatgccaa gttctcaagc
     2161 gaaaaattag aattagtttt tagtgaagag atattgcctt atcttttcca gttaaaaaaa
     2221 ttcataaaat ataatctgga acatgttaag tcttttgaaa acaaatactc tatgaggatt
     2281 tatgagtggt tattaaaaga actaacacaa aagaaaactc acaaggcaaa tatagagatt
     2341 agccttgatg aatttaagtt catgttaatg cttgaaaata actaccatga gtttaaaagg
     2401 cttaaccaat gggttttgaa accaataagt aaagatttaa acacttacag caatatgaaa
     2461 ttggtggttg ataagcgagg ccgcccgact gatacgttga ttttccaagt tgaactagat
     2521 agacaaatgg atctcgtaac cgaacttgag aacaaccaga taaaaatgaa tggtgacaaa
     2581 ataccaacaa ccattacatc agattcctac ctacataacg gactaagaaa aacactacac
     2641 gatgctttaa ctgcaaaaat tcagctcacc agttttgagg caaaattttt gagtgacatg
     2701 caaagtaagt atgatctcaa tggttcgttc tcatggctca cgcaaaaaca acgaaccaca
     2761 ctagagaaca tactggctaa atacggaagg atctgaggtt cttatggctc ttgtatctat
     2821 cagtgaagca tcaagactaa caaacaaaag tagaacaact gttcaccgtt acatatcaaa
     2881 gggaaaactg tccatatgca cagatgaaaa cggtgtaaaa aagatagata catcagagct
     2941 tttacgagtt tttggtgcat tcaaagctgt tcaccatgaa cagatcgaca atgtaactac
     3001 tagagctgat ccttcaactc agcaaaagtt cgatttattc aacaaagcca cgttgtgtct
     3061 caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg
     3121 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct
     3181 tgctcccgtc cgcgcttaaa ctccaacatg gacgctgatt tatatgggta taaatgggct
     3241 cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg
     3301 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     3361 gtccgtctca actggctgac ggagtttatg cctctcccga ccatcaagca ttttatccgt
     3421 actcctgatg atgcgtggtt actcaccacc gcgattcctg ggaaaacagc cttccaggta
     3481 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggccgt gttcctgcgc
     3541 cggttacatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgtgt atttcgtctt
     3601 gctcaggcgc aatcacgcat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     3661 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc acaagctctt gccattctca
     3721 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     3781 aaattaatag gttgtattga tgttggacgg gtcggaatcg cagaccgtta ccaggacctt
     3841 gccattcttt ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     3901 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     3961 tttttctaat aatactagca gaaatcatcc ttagcgaaag ctaaggattt tttttatctg
     4021 attaccgcct ttgagtgagc cccaatgata accccaaggg aagttttagt caaaagcctc
     4081 cggtcggagg cttttgactt tctgctaatc taactaacta acccttagtg actcctgcag
     4141 ttattacgct gcaagggcgt aattttcgtc gttcgctgcg ctagctttgt acagttcatc
     4201 cataccatgc gtgatgcccg ctgcggttac gaactccagc agaaccatat gatcgcgttt
     4261 ctcgttcgga tctttagaca gaacgctttg cgtgctcaga tagtgattgt ctggcagcag
     4321 aacaggacca tcaccgattg gagtgttttg ctggtagtga tcagccagct gcacgctgcc
     4381 atcctccacg ttgtggcgaa ttttaaaatt cgctttaatg ccattttttt gtttatcggc
     4441 ggtgatgtaa acattgtggc tgttaaa
//
