LOCUS       sequence-200487-        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"
     RBS             469..480
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    521..537
                     /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        594..628
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        692..767
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           772..839
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     terminator      1014..1108
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      1600..1822
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             1870..2820
                     /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(3167..3186)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3178..3993
                     /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(3251..3270)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(4092..4123)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(join(4210..4467,1..453))
                     /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 gatgtaaaca ttgtggctgt taaaattgta ttccagctta tggcccagga tattgccgtc
       61 ttctttaaag tcaatgcctt tcagctcaat gcggtttacc agggtatcgc cttcaaattt
      121 cacttccgca cgcgttttgt acgtgccgtc atccttaaag gaaatcgtgc gttcctgcac
      181 atagccttcc ggcatggcgg acttgaagaa gtcatgctgc ttcatatggt ccggataacg
      241 agcaaagcac tgaacaccat aagtcagcgt cgttaccaga gtcggccaag gtaccggcag
      301 tttaccagta gtacagatga acttcagcgt cagtttacca ttagttgcgt caccttcacc
      361 ctcgccacgc acggaaaact tatgaccgtt gacatcacca tccagttcca ccagaatagg
      421 gacgacacca gtgaacagct cttcgccttt acgactagtc atctagtatt tctcctcttt
      481 ctctagtagg tcagtgcgtc ctgctgatgt gctcagtatc ttgttatccg ctcacaatgt
      541 caatgttatc cgctcacatt tattgacgcc aggcaagtct cagctgggtc gacttgacag
      601 ctagctcagt cctaggtata atgctagcgc gacttcatgc tgtaaaggtt ttgcacgttt
      661 ctttgtctgc cgtgcaaaac ctttcgcggt agttttagag ctagaaatag caagttaaaa
      721 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tggccggcat
      781 ggtcccagcc tcctcgctgg cgccggctgg gcaacatgct tcggcatggc gaatgggact
      841 tgttcagaac gctcggtctt gcacaccggg cgttttttct ttgtgagtcc agacgtcagg
      901 tggcaaacgc gaagtaatct tttcggtttt aaagaaaaag ggcagggtgg tgacaccttg
      961 cccgtttttt tgccggagct agagctgtca gaccaagttt acgagctcgc ttggactcct
     1021 gttgatagat ccagtaatga cctcagaact ccatctggat ttgttcagaa cgctcggttg
     1081 ccgccgggcg ttttttattg gtgagaatcc aagcactagg gacagtaaga cgggtaagcc
     1141 tgttgatgat accgctgcct tactgggtgc attagccagt ctgaatgacc tgtcacggga
     1201 taatccgaag tggtcagact ggaaaatcag agggcaggaa ctgctgaaca gcaaaaagtc
     1261 agatagcacc acatagcaga cccgccataa aacgccctga gaagcccgtg acgggctttt
     1321 cttgtattat gggtagtttc cttgcatgaa tccataaaag gcgcctgtag tgccatttac
     1381 ccccattcac tgccagagcc gtgagcgcag cgaactgaat gtcacgaaaa agacagcgac
     1441 tcaggtgcct gatggtcgga gacaaaagga atattcagcg atttgcccga gcttgcgagg
     1501 gtgctactta agcctttagg gttttaaggt ctgttttgta gaggagcaaa cagcgtttgc
     1561 gacatccttt tgtaatactg cggaactgac taaagtagtg agttatacac agggctggga
     1621 tctattcttt ttatcttttt ttattctttc tttattctat aaattataac cacttgaata
     1681 taaacaaaaa aaacacacaa aggtctagcg gaatttacag agggtctagc agaatttaca
     1741 agttttccag caaaggtcta gcagaattta cagataccca caactcaaag gaaaaggaca
     1801 tgtaattatc attgactagc ccatctcaat tggtatagtg attaaaatca cctagaccaa
     1861 ttgagatgta tgtctgaatt agttgttttc aaagcaaatg aactagcgat tagtcgctat
     1921 gacttaacgg agcatgaaac caagctaatt ttatgctgtg tggcactact caaccccacg
     1981 attgaaaacc ctacaaggaa agaacggacg gtatcgttca cttataacca atacgctcag
     2041 atgatgaaca tcagtaggga aaatgcttat ggtgtattag ctaaagcaac cagagagctg
     2101 atgacgagaa ctgtggaaat caggaatcct ttggttaaag gctttgggat tttccagtgg
     2161 acaaactatg ccaagttctc aagcgaaaaa ttagaattag tttttagtga agagatattg
     2221 ccttatcttt tccagttaaa aaaattcata aaatataatc tggaacatgt taagtctttt
     2281 gaaaacaaat actctatgag gatttatgag tggttattaa aagaactaac acaaaagaaa
     2341 actcacaagg caaatataga gattagcctt gatgaattta agttcatgtt aatgcttgaa
     2401 aataactacc atgagtttaa aaggcttaac caatgggttt tgaaaccaat aagtaaagat
     2461 ttaaacactt acagcaatat gaaattggtg gttgataagc gaggccgccc gactgatacg
     2521 ttgattttcc aagttgaact agatagacaa atggatctcg taaccgaact tgagaacaac
     2581 cagataaaaa tgaatggtga caaaatacca acaaccatta catcagattc ctacctacat
     2641 aacggactaa gaaaaacact acacgatgct ttaactgcaa aaattcagct caccagtttt
     2701 gaggcaaaat ttttgagtga catgcaaagt aagtatgatc tcaatggttc gttctcatgg
     2761 ctcacgcaaa aacaacgaac cacactagag aacatactgg ctaaatacgg aaggatctga
     2821 ggttcttatg gctcttgtat ctatcagtga agcatcaaga ctaacaaaca aaagtagaac
     2881 aactgttcac cgttacatat caaagggaaa actgtccata tgcacagatg aaaacggtgt
     2941 aaaaaagata gatacatcag agcttttacg agtttttggt gcattcaaag ctgttcacca
     3001 tgaacagatc gacaatgtaa ctactagagc tgatccttca actcagcaaa agttcgattt
     3061 attcaacaaa gccacgttgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat
     3121 atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg
     3181 agccatattc aacgggaaac gtcttgctcc cgtccgcgct taaactccaa catggacgct
     3241 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     3301 cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     3361 gccaatgatg ttacagatga gatggtccgt ctcaactggc tgacggagtt tatgcctctc
     3421 ccgaccatca agcattttat ccgtactcct gatgatgcgt ggttactcac caccgcgatt
     3481 cctgggaaaa cagccttcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     3541 gatgcgctgg ccgtgttcct gcgccggtta cattcgattc ctgtttgtaa ttgtcctttt
     3601 aacagcgatc gtgtatttcg tcttgctcag gcgcaatcac gcatgaataa cggtttggtt
     3661 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     3721 atgcacaagc tcttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     3781 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgggtcgga
     3841 atcgcagacc gttaccagga ccttgccatt ctttggaact gcctcggtga gttttctcct
     3901 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     3961 cagtttcatt tgatgctcga tgagtttttc taataatact agcagaaatc atccttagcg
     4021 aaagctaagg atttttttta tctgattacc gcctttgagt gagccccaat gataacccca
     4081 agggaagttt tagtcaaaag cctccggtcg gaggcttttg actttctgct aatctaacta
     4141 actaaccctt agtgactcct gcagttatta cgctgcaagg gcgtaatttt cgtcgttcgc
     4201 tgcgctagct ttgtacagtt catccatacc atgcgtgatg cccgctgcgg ttacgaactc
     4261 cagcagaacc atatgatcgc gtttctcgtt cggatcttta gacagaacgc tttgcgtgct
     4321 cagatagtga ttgtctggca gcagaacagg accatcaccg attggagtgt tttgctggta
     4381 gtgatcagcc agctgcacgc tgccatcctc cacgttgtgg cgaattttaa aattcgcttt
     4441 aatgccattt ttttgtttat cggcggt
//
