LOCUS       sequence-372776-        4660 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. Coli RNase III under control of pBAD promoter and 
            constitutively expresses gUTR-129-GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4660)
  AUTHORS   Hauk P, Weeks R, Ostermeier M
  TITLE     A CRISPR-dCas9 System for Assaying and Selecting for RNase III 
            Activity In Vivo in Escherichia coli.
  JOURNAL   CRISPR J. 2022 Dec 9. doi: 10.1089/crispr.2022.0041.
  PUBMED    36493370
REFERENCE   2  (bases 1 to 4660)
  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: "CRISPR J. 
            2022 Dec 9. doi: 10.1089/crispr.2022.0041."
COMMENT     Sequence Label: pRnc-gGFP129
FEATURES             Location/Qualifiers
     source          1..4660
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        16..50
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        200..275
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             296..325
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             341..358
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(405..1283)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1310..1594
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1517..1536
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     primer_bind     complement(2361..2378)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2531..2577
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2630..2647)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2801..3389)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2881..2900)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3560..4351,4352..4420))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4183..4202
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4421..4525)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4593..4611
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(4615..4658)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 gaattctaaa gatctttgac agctagctca gtcctaggta taatactagt gaagtttaag
       61 gttggcacct ccaggttgcc acggcacacg aaacagcgtt ggtccccata taccggtaaa
      121 ctgaaactgc agcgaagcag ttagcagaac catgtatatc aggtctgttt cgtgtgctgc
      181 atctaattca acaagaattg ttttagagct agaaatagca agttaaaata aggctagtcc
      241 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca
      301 aaaactcatc tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg
      361 agtttaaacg gtctccagcc gtcaattgtc tgattcgtta ccaattatga caacttgacg
      421 gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg
      481 cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg
      541 gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc
      601 tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac
      661 ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga
      721 tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg
      781 ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc
      841 gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc
      901 ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg caaatattga cggccagtta
      961 agccattcat gccagtaggc gcgcggacga aagtaaaccc actggtgata ccattcgcga
     1021 gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc
     1081 ggtcggcaaa caaattctcg tccctgattt ttcaccaccc cctgaccgcg aatggtgaga
     1141 ttgagaatat aacctttcat tcccagcggt cggtcgataa aaaaatcgag ataaccgttg
     1201 gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg
     1261 ggatcatttt gcgcttcagc catacttttc atactcccgc cattcagaga agaaaccaat
     1321 tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt actggctctt ctcgctaacc
     1381 aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca
     1441 aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag tccacattga ttatttgcac
     1501 ggcgtcacac tttgctatgc catagcattt ttatccataa gattagcgga tcctacctga
     1561 cgctttttat cgcaactctc tactgtttct ccatacccgt ttttttgggc tagcactagt
     1621 gaaggagata tacatatgaa ccccatcgta attaatcggc ttcaacggaa gctgggctac
     1681 acttttaatc atcaggaact gttgcagcag gcattaactc atcgtagtgc cagcagtaaa
     1741 cataacgagc gtttagaatt tttaggcgac tctattctga gctacgttat cgccaatgcg
     1801 ctttatcacc gtttccctcg tgtggatgaa ggcgatatga gccggatgcg cgccacgctg
     1861 gtccgtggca atacgctggc ggaactggcg cgcgaatttg agttaggcga gtgcttacgt
     1921 ttagggccag gtgaacttaa aagcggtgga tttcgtcgtg agtcaattct cgccgacacc
     1981 gtcgaagcat taattggtgg cgtattcctc gacagtgata ttcaaaccgt cgagaaatta
     2041 atcctcaact ggtatcaaac tcgtttggac gaaattagcc caggcgataa acaaaaagat
     2101 ccgaaaacgc gcttgcaaga atatttgcag ggtcgccatc tgccgctgcc gacttatctg
     2161 gtagtccagg tacgtggcga agcgcacgat caggaattta ctatccactg ccaggtcagc
     2221 ggcctgagtg aaccggtggt tggcacaggt tcaagccgtc gtaaggctga gcaggctgcc
     2281 gccgaacagg cgttgaaaaa actggagctg gaatgagagc tcaagcttgg ctgttttggc
     2341 ggatgagaga agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata
     2401 aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca
     2461 gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac
     2521 tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     2581 ttgtttgtcg gtgaactgga tccttactcg agtctagact gcaggcttcc tcgctcactg
     2641 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2701 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2761 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccacagg ctccgccccc
     2821 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2881 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2941 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3001 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3061 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3121 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3181 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3241 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3301 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3361 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3421 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3481 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3541 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3601 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3661 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     3721 cagatttatc agcaataaac cagccagctg gaagggccga gcgcagaagt ggtcctgcaa
     3781 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3841 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     3901 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3961 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4021 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4081 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4141 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4201 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4261 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4321 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4381 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     4441 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4501 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     4561 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg cagaatttca
     4621 gataaaaaaa atccttagct ttcgctaagg atgatttctg
//
