LOCUS       sequence-489240-        3121 bp    DNA     circular SYN 14-OCT-2025
DEFINITION  Expression of a boxB scRNA J102 and an MS2 scRNA J110.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3121)
  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 3121)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 14, 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: pCK677.102-110
FEATURES             Location/Qualifiers
     source          1..3121
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(60..79)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     670..689
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     922..940
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(944..987)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     promoter        1005..1039
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     protein_bind    1072..1090
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    1127..1145
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     CDS             1186..1215
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1231..1248
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1474..1520
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        1550..1584
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        1668..1686
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     CDS             1709..1738
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1754..1771
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1997..2043
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2096..2113)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2267..2855)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2347..2366)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2945..3039)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(3070..3121,1..743))
                     /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"
ORIGIN
        1 gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca
       61 atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag
      121 tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca
      181 tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc gaacagttcg
      241 gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc
      301 atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc
      361 ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga
      421 gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt
      481 cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac
      541 gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc ggtcttgaca
      601 aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt
      661 gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg
      721 tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcagatcat
      781 gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac tttgcagggc
      841 ttcccaacct taccagaggg cgccccagct ggcaattccg acgtctaaga aaccattatt
      901 atcatgacat taacctataa aaataggcgt atcacgaggc agaatttcag ataaaaaaaa
      961 tccttagctt tcgctaagga tgatttctgg aattctaaag atctttgaca gctagctcag
     1021 tcctaggtat aatactagta ggtatccggt ggaacccaag tttgagagct agggccctga
     1081 agaagggccc tagcaagttc aaataaggct agtccgttat caacttgggc cctgaagaag
     1141 ggcccaagtg gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca aaaactcatc
     1201 tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg
     1261 gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat
     1321 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc
     1381 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt
     1441 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
     1501 gactgggcct ttcgttttat ctgttgtttg tcggtgaact ggatcttact tgacagctag
     1561 ctcagtccta ggtataatac tagtaggaca ccgcaggata cctggtttta gagctagaaa
     1621 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcaca tgaggatcac
     1681 ccatgtgctt tttttgaagc ttgggcccga acaaaaactc atctcagaag aggatctgaa
     1741 tagcgccgtc gaccatcatc atcatcatca ttgagtttaa acggtctcca gcttggctgt
     1801 tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt
     1861 ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg
     1921 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta
     1981 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     2041 tatctgttgt ttgtcggtga actggatcct tactcgagtc tagactgcag gcttcctcgc
     2101 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     2161 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     2221 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cacaggctcc
     2281 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     2341 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     2401 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     2461 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     2521 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     2581 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     2641 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     2701 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     2761 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     2821 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     2881 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agagtactgc
     2941 ttggattctc accaataaaa aacgcccggc ggcaaccgag cgttctgaac aaatccagat
     3001 ggagttctga ggtcattact ggatctatca acaggagtcc aagcgagctc tcgaacccca
     3061 gagtcccgct cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg
     3121 a
//
