LOCUS       sequence-197249-        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"
     ncRNA           complement(143..210)
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     misc_RNA        complement(215..290)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(352..386)
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     protein_bind    443..459
                     /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)."
     RBS             500..511
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             527..1237
                     /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"
     terminator      1324..1355
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(1454..2269)
                     /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     2177..2196
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2261..2280
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(2627..3577)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      3625..3847
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(4339..4433)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 tagctccggc aaaaaaacgg gcaaggtgtc accaccctgc cctttttctt taaaaccgaa
       61 aagattactt cgcgtttgcc acctgacgtc tggactcaca aagaaaaaac gcccggtgtg
      121 caagaccgag cgttctgaac aagtcccatt cgccatgccg aagcatgttg cccagccggc
      181 gccagcgagg aggctgggac catgccggcc aaaagcaccg actcggtgcc actttttcaa
      241 gttgataacg gactagcctt attttaactt gctatttcta gctctaaaac taccgcgaaa
      301 ggttttgcac agactctgct ataaggtgca aaacctttct tattaggaca cgctagcatt
      361 atacctagga ctgagctagc tgtcaagtcg acccagctga gacttgcctg gcgtcaataa
      421 atgtgagcgg ataacattga cattgtgagc ggataacaag atactgagca catcagcagg
      481 acgcactgac ctactagaga aagaggagaa atactagatg actagtcgta aaggcgaaga
      541 gctgttcact ggtgtcgtcc ctattctggt ggaactggat ggtgatgtca acggtcataa
      601 gttttccgtg cgtggcgagg gtgaaggtga cgcaactaat ggtaaactga cgctgaagtt
      661 catctgtact actggtaaac tgccggtacc ttggccgact ctggtaacga cgctgactta
      721 tggtgttcag tgctttgctc gttatccgga ccatatgaag cagcatgact tcttcaagtc
      781 cgccatgccg gaaggctatg tgcaggaacg cacgatttcc tttaaggatg acggcacgta
      841 caaaacgcgt gcggaagtga aatttgaagg cgataccctg gtaaaccgca ttgagctgaa
      901 aggcattgac tttaaagaag acggcaatat cctgggccat aagctggaat acaattttaa
      961 cagccacaat gtttacatca ccgccgataa acaaaaaaat ggcattaaag cgaattttaa
     1021 aattcgccac aacgtggagg atggcagcgt gcagctggct gatcactacc agcaaaacac
     1081 tccaatcggt gatggtcctg ttctgctgcc agacaatcac tatctgagca cgcaaagcgt
     1141 tctgtctaaa gatccgaacg agaaacgcga tcatatggtt ctgctggagt tcgtaaccgc
     1201 agcgggcatc acgcatggta tggatgaact gtacaaagct agcgcagcga acgacgaaaa
     1261 ttacgccctt gcagcgtaat aactgcagga gtcactaagg gttagttagt tagattagca
     1321 gaaagtcaaa agcctccgac cggaggcttt tgactaaaac ttcccttggg gttatcattg
     1381 gggctcactc aaaggcggta atcagataaa aaaaatcctt agctttcgct aaggatgatt
     1441 tctgctagta ttattagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     1501 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     1561 cgaggcagtt ccaaagaatg gcaaggtcct ggtaacggtc tgcgattccg acccgtccaa
     1621 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     1681 catgagtgac gactgaatcc ggtgagaatg gcaagagctt gtgcatttct ttccagactt
     1741 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     1801 tcatgcgtga ttgcgcctga gcaagacgaa atacacgatc gctgttaaaa ggacaattac
     1861 aaacaggaat cgaatgtaac cggcgcagga acacggccag cgcatcaaca atattttcac
     1921 ctgaatcagg atattcttct aatacctgga aggctgtttt cccaggaatc gcggtggtga
     1981 gtaaccacgc atcatcagga gtacggataa aatgcttgat ggtcgggaga ggcataaact
     2041 ccgtcagcca gttgagacgg accatctcat ctgtaacatc attggcaacg ctacctttgc
     2101 catgtttcag aaacaactct ggcgcatcgg gcttcccata caagcgatag attgtcgcac
     2161 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagcg tccatgttgg
     2221 agtttaagcg cggacgggag caagacgttt cccgttgaat atggctcata acaccccttg
     2281 tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg
     2341 caatgtaaca tcagagattt tgagacacaa cgtggctttg ttgaataaat cgaacttttg
     2401 ctgagttgaa ggatcagctc tagtagttac attgtcgatc tgttcatggt gaacagcttt
     2461 aaatgcacca aaaactcgta aaagctctga tgtatctatc ttttttacac cgttttcatc
     2521 tgtgcatatg gacagttttc cctttgatat ctaacggtga acagttgttc tacttttgtt
     2581 tgttagtctt gatgcttcac tgatagatac aagagccata agaacctcag atccttccgt
     2641 atttagccag tatgttctct agtgtggttc gttgtttttg cgtgagccat gagaacgaac
     2701 cattgagatc atgcttactt tgcatgtcac tcaaaaattt tgcctcaaaa ctggtgagct
     2761 gaatttttgc agttaaagca tcgtgtagtg tttttcttag tccgttacgt aggtaggaat
     2821 ctgatgtaat ggttgttggt attttgtcac cattcatttt tatctggttg ttctcaagtt
     2881 cggttacgag atccatttgt ctatctagtt caacttggaa aatcaacgta tcagtcgggc
     2941 ggcctcgctt atcaaccacc aatttcatat tgctgtaagt gtttaaatct ttacttattg
     3001 gtttcaaaac ccattggtta agccttttaa actcatggta gttattttca agcattaaca
     3061 tgaacttaaa ttcatcaagg ctaatctcta tatttgcctt gtgagttttc ttttgtgtta
     3121 gttcttttaa taaccactca taaatcctca tagagtattt gttttcaaaa gacttaacat
     3181 gttccagatt atattttatg aattttttta actggaaaag ataaggcaat atctcttcac
     3241 taaaaactaa ttctaatttt tcgcttgaga acttggcata gtttgtccac tggaaaatct
     3301 caaagccttt aaccaaagga ttcctgattt ccacagttct cgtcatcagc tctctggttg
     3361 ctttagctaa tacaccataa gcattttccc tactgatgtt catcatctga gcgtattggt
     3421 tataagtgaa cgataccgtc cgttctttcc ttgtagggtt ttcaatcgtg gggttgagta
     3481 gtgccacaca gcataaaatt agcttggttt catgctccgt taagtcatag cgactaatcg
     3541 ctagttcatt tgctttgaaa acaactaatt cagacataca tctcaattgg tctaggtgat
     3601 tttaatcact ataccaattg agatgggcta gtcaatgata attacatgtc cttttccttt
     3661 gagttgtggg tatctgtaaa ttctgctaga cctttgctgg aaaacttgta aattctgcta
     3721 gaccctctgt aaattccgct agacctttgt gtgttttttt tgtttatatt caagtggtta
     3781 taatttatag aataaagaaa gaataaaaaa agataaaaag aatagatccc agccctgtgt
     3841 ataactcact actttagtca gttccgcagt attacaaaag gatgtcgcaa acgctgtttg
     3901 ctcctctaca aaacagacct taaaacccta aaggcttaag tagcaccctc gcaagctcgg
     3961 gcaaatcgct gaatattcct tttgtctccg accatcaggc acctgagtcg ctgtcttttt
     4021 cgtgacattc agttcgctgc gctcacggct ctggcagtga atgggggtaa atggcactac
     4081 aggcgccttt tatggattca tgcaaggaaa ctacccataa tacaagaaaa gcccgtcacg
     4141 ggcttctcag ggcgttttat ggcgggtctg ctatgtggtg ctatctgact ttttgctgtt
     4201 cagcagttcc tgccctctga ttttccagtc tgaccacttc ggattatccc gtgacaggtc
     4261 attcagactg gctaatgcac ccagtaaggc agcggtatca tcaacaggct tacccgtctt
     4321 actgtcccta gtgcttggat tctcaccaat aaaaaacgcc cggcggcaac cgagcgttct
     4381 gaacaaatcc agatggagtt ctgaggtcat tactggatct atcaacagga gtccaagcga
     4441 gctcgtaaac ttggtctgac agctc
//
