LOCUS       sequence-197247-        4467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3A 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: PAJ379
FEATURES             Location/Qualifiers
     source          1..4467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      33..127
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      619..841
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             889..1839
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     primer_bind     complement(2186..2205)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2197..3012
                     /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(2270..2289)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(3111..3142)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(3229..3939)
                     /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"
     RBS             3955..3966
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    4007..4023
                     /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        4080..4114
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        4178..4253
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           4258..4325
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
ORIGIN
        1 gagctgtcag accaagttta cgagctcgct tggactcctg ttgatagatc cagtaatgac
       61 ctcagaactc catctggatt tgttcagaac gctcggttgc cgccgggcgt tttttattgg
      121 tgagaatcca agcactaggg acagtaagac gggtaagcct gttgatgata ccgctgcctt
      181 actgggtgca ttagccagtc tgaatgacct gtcacgggat aatccgaagt ggtcagactg
      241 gaaaatcaga gggcaggaac tgctgaacag caaaaagtca gatagcacca catagcagac
      301 ccgccataaa acgccctgag aagcccgtga cgggcttttc ttgtattatg ggtagtttcc
      361 ttgcatgaat ccataaaagg cgcctgtagt gccatttacc cccattcact gccagagccg
      421 tgagcgcagc gaactgaatg tcacgaaaaa gacagcgact caggtgcctg atggtcggag
      481 acaaaaggaa tattcagcga tttgcccgag cttgcgaggg tgctacttaa gcctttaggg
      541 ttttaaggtc tgttttgtag aggagcaaac agcgtttgcg acatcctttt gtaatactgc
      601 ggaactgact aaagtagtga gttatacaca gggctgggat ctattctttt tatctttttt
      661 tattctttct ttattctata aattataacc acttgaatat aaacaaaaaa aacacacaaa
      721 ggtctagcgg aatttacaga gggtctagca gaatttacaa gttttccagc aaaggtctag
      781 cagaatttac agatacccac aactcaaagg aaaaggacat gtaattatca ttgactagcc
      841 catctcaatt ggtatagtga ttaaaatcac ctagaccaat tgagatgtat gtctgaatta
      901 gttgttttca aagcaaatga actagcgatt agtcgctatg acttaacgga gcatgaaacc
      961 aagctaattt tatgctgtgt ggcactactc aaccccacga ttgaaaaccc tacaaggaaa
     1021 gaacggacgg tatcgttcac ttataaccaa tacgctcaga tgatgaacat cagtagggaa
     1081 aatgcttatg gtgtattagc taaagcaacc agagagctga tgacgagaac tgtggaaatc
     1141 aggaatcctt tggttaaagg ctttgagatt ttccagtgga caaactatgc caagttctca
     1201 agcgaaaaat tagaattagt ttttagtgaa gagatattgc cttatctttt ccagttaaaa
     1261 aaattcataa aatataatct ggaacatgtt aagtcttttg aaaacaaata ctctatgagg
     1321 atttatgagt ggttattaaa agaactaaca caaaagaaaa ctcacaaggc aaatatagag
     1381 attagccttg atgaatttaa gttcatgtta atgcttgaaa ataactacca tgagtttaaa
     1441 aggcttaacc aatgggtttt gaaaccaata agtaaagatt taaacactta cagcaatatg
     1501 aaattggtgg ttgataagcg aggccgcccg actgatacgt tgattttcca agttgaacta
     1561 gatagacaaa tggatctcgt aaccgaactt gagaacaacc agataaaaat gaatggtgac
     1621 aaaataccaa caaccattac atcagattcc tacctacgta acggactaag aaaaacacta
     1681 cacgatgctt taactgcaaa aattcagctc accagttttg aggcaaaatt tttgagtgac
     1741 atgcaaagta agcatgatct caatggttcg ttctcatggc tcacgcaaaa acaacgaacc
     1801 acactagaga acatactggc taaatacgga aggatctgag gttcttatgg ctcttgtatc
     1861 tatcagtgaa gcatcaagac taacaaacaa aagtagaaca actgttcacc gttagatatc
     1921 aaagggaaaa ctgtccatat gcacagatga aaacggtgta aaaaagatag atacatcaga
     1981 gcttttacga gtttttggtg catttaaagc tgttcaccat gaacagatcg acaatgtaac
     2041 tactagagct gatccttcaa ctcagcaaaa gttcgattta ttcaacaaag ccacgttgtg
     2101 tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg aacaataaaa
     2161 ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg
     2221 tcttgctccc gtccgcgctt aaactccaac atggacgctg atttatatgg gtataaatgg
     2281 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg gaagcccgat
     2341 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     2401 atggtccgtc tcaactggct gacggagttt atgcctctcc cgaccatcaa gcattttatc
     2461 cgtactcctg atgatgcgtg gttactcacc accgcgattc ctgggaaaac agccttccag
     2521 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc cgtgttcctg
     2581 cgccggttac attcgattcc tgtttgtaat tgtcctttta acagcgatcg tgtatttcgt
     2641 cttgctcagg cgcaatcacg catgaataac ggtttggttg atgcgagtga ttttgatgac
     2701 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcacaagct cttgccattc
     2761 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     2821 gggaaattaa taggttgtat tgatgttgga cgggtcggaa tcgcagaccg ttaccaggac
     2881 cttgccattc tttggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     2941 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
     3001 gagtttttct aataatacta gcagaaatca tccttagcga aagctaagga ttttttttat
     3061 ctgattaccg cctttgagtg agccccaatg ataaccccaa gggaagtttt agtcaaaagc
     3121 ctccggtcgg aggcttttga ctttctgcta atctaactaa ctaaccctta gtgactcctg
     3181 cagttattac gctgcaaggg cgtaattttc gtcgttcgct gcgctagctt tgtacagttc
     3241 atccatacca tgcgtgatgc ccgctgcggt tacgaactcc agcagaacca tatgatcgcg
     3301 tttctcgttc ggatctttag acagaacgct ttgcgtgctc agatagtgat tgtctggcag
     3361 cagaacagga ccatcaccga ttggagtgtt ttgctggtag tgatcagcca gctgcacgct
     3421 gccatcctcc acgttgtggc gaattttaaa attcgcttta atgccatttt tttgtttatc
     3481 ggcggtgatg taaacattgt ggctgttaaa attgtattcc agcttatggc ccaggatatt
     3541 gccgtcttct ttaaagtcaa tgcctttcag ctcaatgcgg tttaccaggg tatcgccttc
     3601 aaatttcact tccgcacgcg ttttgtacgt gccgtcatcc ttaaaggaaa tcgtgcgttc
     3661 ctgcacatag ccttccggca tggcggactt gaagaagtca tgctgcttca tatggtccgg
     3721 ataacgagca aagcactgaa caccataagt cagcgtcgtt accagagtcg gccaaggtac
     3781 cggcagttta ccagtagtac agatgaactt cagcgtcagt ttaccattag ttgcgtcacc
     3841 ttcaccctcg ccacgcacgg aaaacttatg accgttgaca tcaccatcca gttccaccag
     3901 aatagggacg acaccagtga acagctcttc gcctttacga ctagtcatct agtatttctc
     3961 ctctttctct agtaggtcag tgcgtcctgc tgatgtgctc agtatcttgt tatccgctca
     4021 caatgtcaat gttatccgct cacatttatt gacgccaggc aagtctcagc tgggtcgact
     4081 tgacagctag ctcagtccta ggtataatgc tagcgcgact tcatgctgta aaggttttgc
     4141 acgtttcttt gtctgccgtg caaaaccttt cgcggtagtt ttagagctag aaatagcaag
     4201 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttggc
     4261 cggcatggtc ccagcctcct cgctggcgcc ggctgggcaa catgcttcgg catggcgaat
     4321 gggacttgtt cagaacgctc ggtcttgcac accgggcgtt ttttctttgt gagtccagac
     4381 gtcaggtggc aaacgcgaag taatcttttc ggttttaaag aaaaagggca gggtggtgac
     4441 accttgcccg tttttttgcc ggagcta
//
