LOCUS       sequence-200483-        4465 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses HIV3 igRNA and sfGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4465)
  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 4465)
  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: PAJ426
FEATURES             Location/Qualifiers
     source          1..4465
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             427..438
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    479..495
                     /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        552..586
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        648..723
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           728..795
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     terminator      970..1064
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      1556..1778
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             1826..2776
                     /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(3123..3142)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3134..3949
                     /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(3207..3226)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(4048..4079)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(join(4166..4465,1..411))
                     /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 gcccaggata ttgccgtctt ctttaaagtc aatgcctttc agctcaatgc ggtttaccag
       61 ggtatcgcct tcaaatttca cttccgcacg cgttttgtac gtgccgtcat ccttaaagga
      121 aatcgtgcgt tcctgcacat agccttccgg catggcggac ttgaagaagt catgctgctt
      181 catatggtcc ggataacgag caaagcactg aacaccataa gtcagcgtcg ttaccagagt
      241 cggccaaggt accggcagtt taccagtagt acagatgaac ttcagcgtca gtttaccatt
      301 agttgcgtca ccttcaccct cgccacgcac ggaaaactta tgaccgttga catcaccatc
      361 cagttccacc agaataggga cgacaccagt gaacagctct tcgcctttac gactagtcat
      421 ctagtatttc tcctctttct ctagtaggtc agtgcgtcct gctgatgtgc tcagtatctt
      481 gttatccgct cacaatgtca atgttatccg ctcacattta ttgacgccag gcaagtctca
      541 gctgggtcga cttgacagct agctcagtcc taggtataat gctagcgtgt cctaataaga
      601 aaggttttgc accttatagc agagtctgtg caaaaccttt cgcggtagtt ttagagctag
      661 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
      721 tgcttttggc cggcatggtc ccagcctcct cgctggcgcc ggctgggcaa catgcttcgg
      781 catggcgaat gggacttgtt cagaacgctc ggtcttgcac accgggcgtt ttttctttgt
      841 gagtccagac gtcaggtggc aaacgcgaag taatcttttc ggttttaaag aaaaagggca
      901 gggtggtgac accttgcccg tttttttgcc ggagctagag ctgtcagacc aagtttacga
      961 gctcgcttgg actcctgttg atagatccag taatgacctc agaactccat ctggatttgt
     1021 tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaagc actagggaca
     1081 gtaagacggg taagcctgtt gatgataccg ctgccttact gggtgcatta gccagtctga
     1141 atgacctgtc acgggataat ccgaagtggt cagactggaa aatcagaggg caggaactgc
     1201 tgaacagcaa aaagtcagat agcaccacat agcagacccg ccataaaacg ccctgagaag
     1261 cccgtgacgg gcttttcttg tattatgggt agtttccttg catgaatcca taaaaggcgc
     1321 ctgtagtgcc atttaccccc attcactgcc agagccgtga gcgcagcgaa ctgaatgtca
     1381 cgaaaaagac agcgactcag gtgcctgatg gtcggagaca aaaggaatat tcagcgattt
     1441 gcccgagctt gcgagggtgc tacttaagcc tttagggttt taaggtctgt tttgtagagg
     1501 agcaaacagc gtttgcgaca tccttttgta atactgcgga actgactaaa gtagtgagtt
     1561 atacacaggg ctgggatcta ttctttttat ctttttttat tctttcttta ttctataaat
     1621 tataaccact tgaatataaa caaaaaaaac acacaaaggt ctagcggaat ttacagaggg
     1681 tctagcagaa tttacaagtt ttccagcaaa ggtctagcag aatttacaga tacccacaac
     1741 tcaaaggaaa aggacatgta attatcattg actagcccat ctcaattggt atagtgatta
     1801 aaatcaccta gaccaattga gatgtatgtc tgaattagtt gttttcaaag caaatgaact
     1861 agcgattagt cgctatgact taacggagca tgaaaccaag ctaattttat gctgtgtggc
     1921 actactcaac cccacgattg aaaaccctac aaggaaagaa cggacggtat cgttcactta
     1981 taaccaatac gctcagatga tgaacatcag tagggaaaat gcttatggtg tattagctaa
     2041 agcaaccaga gagctgatga cgagaactgt ggaaatcagg aatcctttgg ttaaaggctt
     2101 tgggattttc cagtggacaa actatgccaa gttctcaagc gaaaaattag aattagtttt
     2161 tagtgaagag atattgcctt atcttttcca gttaaaaaaa ttcataaaat ataatctgga
     2221 acatgttaag tcttttgaaa acaaatactc tatgaggatt tatgagtggt tattaaaaga
     2281 actaacacaa aagaaaactc acaaggcaaa tatagagatt agccttgatg aatttaagtt
     2341 catgttaatg cttgaaaata actaccatga gtttaaaagg cttaaccaat gggttttgaa
     2401 accaataagt aaagatttaa acacttacag caatatgaaa ttggtggttg ataagcgagg
     2461 ccgcccgact gatacgttga ttttccaagt tgaactagat agacaaatgg atctcgtaac
     2521 cgaacttgag aacaaccaga taaaaatgaa tggtgacaaa ataccaacaa ccattacatc
     2581 agattcctac ctacataacg gactaagaaa aacactacac gatgctttaa ctgcaaaaat
     2641 tcagctcacc agttttgagg caaaattttt gagtgacatg caaagtaagt atgatctcaa
     2701 tggttcgttc tcatggctca cgcaaaaaca acgaaccaca ctagagaaca tactggctaa
     2761 atacggaagg atctgaggtt cttatggctc ttgtatctat cagtgaagca tcaagactaa
     2821 caaacaaaag tagaacaact gttcaccgtt acatatcaaa gggaaaactg tccatatgca
     2881 cagatgaaaa cggtgtaaaa aagatagata catcagagct tttacgagtt tttggtgcat
     2941 tcaaagctgt tcaccatgaa cagatcgaca atgtaactac tagagctgat ccttcaactc
     3001 agcaaaagtt cgatttattc aacaaagcca cgttgtgtct caaaatctct gatgttacat
     3061 tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa
     3121 tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctcccgtc cgcgcttaaa
     3181 ctccaacatg gacgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     3241 aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     3301 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtccgtctca actggctgac
     3361 ggagtttatg cctctcccga ccatcaagca ttttatccgt actcctgatg atgcgtggtt
     3421 actcaccacc gcgattcctg ggaaaacagc cttccaggta ttagaagaat atcctgattc
     3481 aggtgaaaat attgttgatg cgctggccgt gttcctgcgc cggttacatt cgattcctgt
     3541 ttgtaattgt ccttttaaca gcgatcgtgt atttcgtctt gctcaggcgc aatcacgcat
     3601 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     3661 acaagtctgg aaagaaatgc acaagctctt gccattctca ccggattcag tcgtcactca
     3721 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     3781 tgttggacgg gtcggaatcg cagaccgtta ccaggacctt gccattcttt ggaactgcct
     3841 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     3901 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat aatactagca
     3961 gaaatcatcc ttagcgaaag ctaaggattt tttttatctg attaccgcct ttgagtgagc
     4021 cccaatgata accccaaggg aagttttagt caaaagcctc cggtcggagg cttttgactt
     4081 tctgctaatc taactaacta acccttagtg actcctgcag ttattacgct gcaagggcgt
     4141 aattttcgtc gttcgctgcg ctagctttgt acagttcatc cataccatgc gtgatgcccg
     4201 ctgcggttac gaactccagc agaaccatat gatcgcgttt ctcgttcgga tctttagaca
     4261 gaacgctttg cgtgctcaga tagtgattgt ctggcagcag aacaggacca tcaccgattg
     4321 gagtgttttg ctggtagtga tcagccagct gcacgctgcc atcctccacg ttgtggcgaa
     4381 ttttaaaatt cgctttaatg ccattttttt gtttatcggc ggtgatgtaa acattgtggc
     4441 tgttaaaatt gtattccagc ttatg
//
