LOCUS       sequence-345702-        4319 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RFP reporter used to evaluate CRISPRa in bacteria, contains gRNA 
            target sites every 10 bp upstream of the promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4319)
  AUTHORS   Villegas Kcam MC, Tsong AJ, Chappell J
  TITLE     Rational engineering of a modular bacterial CRISPR-Cas activation 
            platform with expanded target range.
  JOURNAL   Nucleic Acids Res. 2021 May 7;49(8):4793-4802. doi: 
            10.1093/nar/gkab211.
  PUBMED    33823546
REFERENCE   2  (bases 1 to 4319)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2021-05-7- 7"; volume: "49"; issue: "8";
            pages: "4793-4802"
COMMENT     Sequence Label: pJEC581
FEATURES             Location/Qualifiers
     source          1..4319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(987..1802)
                     /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"
     CDS             complement(987..1802)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /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     1710..1729
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1794..1813
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        2163..2197
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     RBS             2207..2218
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2225..2902
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      2935..3006
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3022..3049
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(join(3580..4319,1..211))
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
ORIGIN
        1 ctaatacacc ataagcattt tccctactga tgttcatcat ctgagcgtat tggttataag
       61 tgaacgatac cgtccgttct ttccttgtag ggttttcaat cgtggggttg agtagtgcca
      121 cacagcataa aattagcttg gtttcatgct ccgttaagtc atagcgacta atcgctagtt
      181 catttgcttt gaaaacaact aattcagaca tacatctcaa ttggtctagg tgattttaat
      241 cactatacca attgagatgg gctagtcaat gataattact agtccttttc ccgggtgatc
      301 tgggtatctg taaattctgc tagacctttg ctggaaaact tgtaaattct gctagaccct
      361 ctgtaaattc cgctagacct ttgtgtgttt tttttgttta tattcaagtg gttataattt
      421 atagaataaa gaaagaataa aaaaagataa aaagaataga tcccagccct gtgtataact
      481 cactacttta gtcagttccg cagtattaca aaaggatgtc gcaaacgctg tttgctcctc
      541 tacaaaacag accttaaaac cctaaaggct taagtagcac cctcgcaagc tcgggcaaat
      601 cgctgaatat tccttttgtc tccgaccatc aggcacctga gtcgctgtct ttttcgtgac
      661 attcagttcg ctgcgctcac ggctctggca gtgaatgggg gtaaatggca ctacaggcgc
      721 cttttatgga ttcatgcaag gaaactaccc ataatacaag aaaagcccgt cacgggcttc
      781 tcagggcgtt ttatggcggg tctgctatgt ggtgctatct gactttttgc tgttcagcag
      841 ttcctgccct ctgattttcc agtctgacca cttcggatta tcccgtgaca ggtcattcag
      901 actggctaat gcacccagta aggcagcggt atcatcaaca ggcttacccg tcttactgtg
      961 tgttacaacc aattaaccaa ttctgattag aaaaactcat cgagcatcaa atgaaactgc
     1021 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     1081 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     1141 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     1201 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag cttatgcatt
     1261 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     1321 accaaaccgt tattcattcg tgattgcgcc tgagcgaggc gaaatacgcg atcgctgtta
     1381 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     1441 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     1501 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     1561 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     1621 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     1681 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     1741 gcatccatgt tggaatttaa tcgcggcctg gagcaagacg tttcccgttg aatatggctc
     1801 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgatgatata
     1861 tttttatctt gtgcaatgta acatcagaga ttttgagaca caacgtggct ttgttgaata
     1921 aatcgaactt ttgctgagtt gaaggatcag cacatttccc cgaaaagtgc cacctgtggc
     1981 aattccgacg tcgcctacgg tatccaccgg agacctatgg cagcctccgg ccgccatagg
     2041 acacctttgg ttgccaaggg tgacctatgg tgaccatggg ccaccacggg cgacctcagg
     2101 tatcctgcgg tgtcctgcgg ttaccaaagg cgtcctttgg gttccaccgg atacctccgg
     2161 acttgacagc tagctcagtc ctagggattg tgctagcgaa ttcattaaag aggagaaagg
     2221 taccatggcg agtagcgaag acgttatcaa agagttcatg cgtttcaaag ttcgtatgga
     2281 aggttccgtt aacggtcacg agttcgaaat cgaaggtgaa ggtgaaggtc gtccgtacga
     2341 aggtacccag accgctaaac tgaaagttac caaaggtggt ccgctgccgt tcgcttggga
     2401 catcctgtcc ccgcagttcc agtacggttc caaagcttac gttaaacacc cggctgacat
     2461 cccggactac ctgaaactgt ccttcccgga aggtttcaaa tgggaacgtg ttatgaactt
     2521 cgaagacggt ggtgttgtta ccgttaccca ggactcctcc ctgcaagacg gtgagttcat
     2581 ctacaaagtt aaactgcgtg gtaccaactt cccgtccgac ggtccggtta tgcagaaaaa
     2641 aaccatgggt tgggaagctt ccaccgaacg tatgtacccg gaagacggtg ctctgaaagg
     2701 tgaaatcaaa atgcgtctga aactgaaaga cggtggtcac tacgacgctg aagttaaaac
     2761 cacctacatg gctaaaaaac cggttcagct gccgggtgct tacaaaaccg acatcaaact
     2821 ggacatcacc tcccacaacg aagactacac catcgttgaa cagtacgaac gtgctgaagg
     2881 tcgtcactcc accggtgctt aaggatccaa actcgagtaa ggatctccag gcatcaaata
     2941 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     3001 gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttatacctag
     3061 ggtacgggtt ttgctgcccg caaacgggct gttctggtgt tgctagtttg ttatcagaat
     3121 cgcagatccg gcttcagccg gtttgccggc tgaaagcgct atttcttcca gaattgccat
     3181 gattttttcc ccacgggagg cgtcactggc tcccgtgttg tcggcagctt tgattcgata
     3241 agcagcatcg cctgtttcag gctgtctatg tgtgactgtt gagctgtaac aagttgtctc
     3301 aggtgttcaa tttcatgttc tagttgcttt gttttactgg tttcacctgt tctattaggt
     3361 gttacatgct gttcatctgt tacattgtcg atctgttcat ggtgaacagc tttgaatgca
     3421 ccaaaaactc gtaaaagctc tgatgtatct atctttttta caccgttttc atctgtgcat
     3481 atggacagtt ttccctttga tatgtaacgg tgaacagttg ttctactttt gtttgttagt
     3541 cttgatgctt cactgataga tacaagagcc ataagaacct cagatccttc cgtatttagc
     3601 cagtatgttc tctagtgtgg ttcgttgttt ttgcgtgagc catgagaacg aaccattgag
     3661 atcatactta ctttgcatgt cactcaaaaa ttttgcctca aaactggtga gctgaatttt
     3721 tgcagttaaa gcatcgtgta gtgtttttct tagtccgtta tgtaggtagg aatctgatgt
     3781 aatggttgtt ggtattttgt caccattcat ttttatctgg ttgttctcaa gttcggttac
     3841 gagatccatt tgtctatcta gttcaacttg gaaaatcaac gtatcagtcg ggcggcctcg
     3901 cttatcaacc accaatttca tattgctgta agtgtttaaa tctttactta ttggtttcaa
     3961 aacccattgg ttaagccttt taaactcatg gtagttattt tcaagcatta acatgaactt
     4021 aaattcatca aggctaatct ctatatttgc cttgtgagtt ttcttttgtg ttagttcttt
     4081 taataaccac tcataaatcc tcatagagta tttgttttca aaagacttaa catgttccag
     4141 attatatttt atgaattttt ttaactggaa aagataaggc aatatctctt cactaaaaac
     4201 taattctaat ttttcgcttg agaacttggc atagtttgtc cactggaaaa tctcaaagcc
     4261 tttaaccaaa ggattcctga tttccacagt tctcgtcatc agctctctgg ttgctttag
//
