LOCUS       sequence-489265-        3081 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a Sth1-dCas9 compatible MS2 scRNA J306 and a Spy-dCas9
            compatible sgRNA J107.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3081)
  AUTHORS   Kiattisewee C, Karanjia AV, Cardiff RAL, Olander KE, Leejareon P, 
            Alvi SS, Carothers JM, Zalatan JG
  TITLE     Systematic Mapping of Bacterial CRISPRa Systems for Synergistic Gene
            Activation Reveals Antagonistic Effects.
  JOURNAL   ACS Synth Biol. 2025 Jul 22. doi: 10.1021/acssynbio.5c00358.
  PUBMED    40693287
REFERENCE   2  (bases 1 to 3081)
  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"; journalName: "ACS Synth 
            Biol. 2025 Jul 22. doi: 10.1021/acssynbio.5c00358."
COMMENT     Sequence Label: pKO024.306-107
FEATURES             Location/Qualifiers
     source          1..3081
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(90..184)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(215..1009)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(326..345)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     936..955
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1188..1206
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(1210..1253)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     promoter        1271..1305
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        1379..1397
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     CDS             1420..1449
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1465..1482
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1708..1754
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        1784..1818
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        1839..1914
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1935..1964
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1980..1997
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2223..2269
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2322..2339)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2493..3081)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2573..2592)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagagt actgcttgga ttctcaccaa taaaaaacgc ccggcggcaa
      121 ccgagcgttc tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg
      181 agtccaagcg agctctcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat
      241 agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag
      301 cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc
      361 ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca
      421 tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc
      481 gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat
      541 catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg
      601 cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag
      661 ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca
      721 cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc
      781 aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca
      841 gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga
      901 acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct
      961 ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc
     1021 ctcatcctgt ctcttgatca gatcatgatc ccctgcgcca tcagatcctt ggcggcaaga
     1081 aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca
     1141 attccgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     1201 cgaggcagaa tttcagataa aaaaaatcct tagctttcgc taaggatgat ttctggaatt
     1261 ctaaagatct ttgacagcta gctcagtcct aggtataata ctagtttgtg tccagaacgc
     1321 tccgtgtttt tgtactcgaa agaagctaca aagataaggc ttcatgccga aatcgcgcac
     1381 atgaggatca cccatgtgct ttttttgaag cttgggcccg aacaaaaact catctcagaa
     1441 gaggatctga atagcgccgt cgaccatcat catcatcatc attgagttta aacggtctcc
     1501 agcttggctg ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac
     1561 gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg
     1621 accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc
     1681 atgctagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg
     1741 gcctttcgtt ttatctgttg tttgtcggtg aactggatct tacttgacag ctagctcagt
     1801 cctaggtata atactagtcg gtgtcctgcg gttaccaagt tttagagcta gaaatagcaa
     1861 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     1921 tgaagcttgg gcccgaacaa aaactcatct cagaagagga tctgaatagc gccgtcgacc
     1981 atcatcatca tcatcattga gtttaaacgg tctccagctt ggctgttttg gcggatgaga
     2041 gaagattttc agcctgatac agattaaatc agaacgcaga agcggtctga taaaacagaa
     2101 tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa
     2161 acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc
     2221 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     2281 cggtgaactg gatccttact cgagtctaga ctgcaggctt cctcgctcac tgactcgctg
     2341 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     2401 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     2461 aggaaccgta aaaaggccgc gttgctggcg tttttccaca ggctccgccc ccctgacgag
     2521 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2581 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2641 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     2701 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2761 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     2821 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     2881 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     2941 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3001 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3061 cgcagaaaaa aaggatctca a
//
