LOCUS       sequence-372775-        4692 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. Coli RNase III under control of pBAD promoter and 
            constitutively expresses gUTR-161-GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4692)
  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 4692)
  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-gGFP161
FEATURES             Location/Qualifiers
     source          1..4692
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     621..640
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(859..963)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1031..1049
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(1053..1096)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     promoter        1114..1148
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        1330..1405
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1426..1455
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1471..1488
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1535..2413)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        2440..2724
                     /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     2647..2666
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     primer_bind     complement(3491..3508)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3661..3707
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3760..3777)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3931..4519)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4011..4030)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4690..4692,1..789,790..858))
                     /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"
ORIGIN
        1 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
       61 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
      121 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
      181 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
      241 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
      301 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
      361 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
      421 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
      481 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
      541 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
      601 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
      661 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
      721 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
      781 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
      841 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
      901 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
      961 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     1021 aacctataaa aataggcgta tcacgaggca gaatttcaga taaaaaaaat ccttagcttt
     1081 cgctaaggat gatttctgga attctaaaga tctttgacag ctagctcagt cctaggtata
     1141 atactagtga agtttaaggt tggcacctcc aggttgccac ggcacacgaa acagcgttgg
     1201 tccccatata ccggtaaact gaaactgcag cgaagcagtt agcagaacca tgtatatcag
     1261 gtctgtttcg tgtgctgaat tgttgacgca tttatttatt ggtatcgcac atctaattca
     1321 acaagaattg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     1381 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca aaaactcatc
     1441 tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg
     1501 gtctccagcc gtcaattgtc tgattcgtta ccaattatga caacttgacg gctacatcat
     1561 tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg cattttttaa
     1621 atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag
     1681 gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc tcgcgccagc
     1741 ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac ggcgacaagc
     1801 aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt
     1861 actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg ttaatcgctt
     1921 ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc gaatagcgcc
     1981 cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg
     2041 cttcatccgg gcgaaagaac cccgtattgg caaatattga cggccagtta agccattcat
     2101 gccagtaggc gcgcggacga aagtaaaccc actggtgata ccattcgcga gcctccggat
     2161 gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa
     2221 caaattctcg tccctgattt ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat
     2281 aacctttcat tcccagcggt cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg
     2341 gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg ggatcatttt
     2401 gcgcttcagc catacttttc atactcccgc cattcagaga agaaaccaat tgtccatatt
     2461 gcatcagaca ttgccgtcac tgcgtctttt actggctctt ctcgctaacc aaaccggtaa
     2521 ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta
     2581 acaaaagtgt ctataatcac ggcagaaaag tccacattga ttatttgcac ggcgtcacac
     2641 tttgctatgc catagcattt ttatccataa gattagcgga tcctacctga cgctttttat
     2701 cgcaactctc tactgtttct ccatacccgt ttttttgggc tagcactagt gaaggagata
     2761 tacatatgaa ccccatcgta attaatcggc ttcaacggaa gctgggctac acttttaatc
     2821 atcaggaact gttgcagcag gcattaactc atcgtagtgc cagcagtaaa cataacgagc
     2881 gtttagaatt tttaggcgac tctattctga gctacgttat cgccaatgcg ctttatcacc
     2941 gtttccctcg tgtggatgaa ggcgatatga gccggatgcg cgccacgctg gtccgtggca
     3001 atacgctggc ggaactggcg cgcgaatttg agttaggcga gtgcttacgt ttagggccag
     3061 gtgaacttaa aagcggtgga tttcgtcgtg agtcaattct cgccgacacc gtcgaagcat
     3121 taattggtgg cgtattcctc gacagtgata ttcaaaccgt cgagaaatta atcctcaact
     3181 ggtatcaaac tcgtttggac gaaattagcc caggcgataa acaaaaagat ccgaaaacgc
     3241 gcttgcaaga atatttgcag ggtcgccatc tgccgctgcc gacttatctg gtagtccagg
     3301 tacgtggcga agcgcacgat caggaattta ctatccactg ccaggtcagc ggcctgagtg
     3361 aaccggtggt tggcacaggt tcaagccgtc gtaaggctga gcaggctgcc gccgaacagg
     3421 cgttgaaaaa actggagctg gaatgagagc tcaagcttgg ctgttttggc ggatgagaga
     3481 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt
     3541 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac
     3601 gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat
     3661 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     3721 gtgaactgga tccttactcg agtctagact gcaggcttcc tcgctcactg actcgctgcg
     3781 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     3841 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3901 gaaccgtaaa aaggccgcgt tgctggcgtt tttccacagg ctccgccccc ctgacgagca
     3961 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4021 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4081 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4141 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4201 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4261 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4321 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4381 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4441 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4501 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4561 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4621 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4681 gtctgacagt ta
//
