LOCUS       sequence-338311-        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(126..941)
                     /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(126..941)
                     /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     849..868
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     933..952
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        1302..1336
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     RBS             1346..1357
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1364..2041
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      2074..2145
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2161..2188
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2719..3669)
                     /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 gtctgaccac ttcggattat cccgtgacag gtcattcaga ctggctaatg cacccagtaa
       61 ggcagcggta tcatcaacag gcttacccgt cttactgtgt gttacaacca attaaccaat
      121 tctgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
      181 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
      241 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
      301 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
      361 acgactgaat ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca
      421 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
      481 gattgcgcct gagcgaggcg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
      541 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
      601 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
      661 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
      721 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
      781 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
      841 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
      901 cgcggcctgg agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
      961 tttatgtaag cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa
     1021 catcagagat tttgagacac aacgtggctt tgttgaataa atcgaacttt tgctgagttg
     1081 aaggatcagc acatttcccc gaaaagtgcc acctgtggca attccgacgt cgcctacggt
     1141 atccaccgga gacctatggc agcctccggc cgccatagga cacctttggt tgccaagggt
     1201 gacctatggt gaccatgggc caccacgggc gacctcaggt atcctgcggt gtcctgcggt
     1261 taccaaaggc gtcctttggg ttccaccgga tacctccgga cttgacagct agctcagtcc
     1321 tagggattgt gctagcgaat tcattaaaga ggagaaaggt accatggcga gtagcgaaga
     1381 cgttatcaaa gagttcatgc gtttcaaagt tcgtatggaa ggttccgtta acggtcacga
     1441 gttcgaaatc gaaggtgaag gtgaaggtcg tccgtacgaa ggtacccaga ccgctaaact
     1501 gaaagttacc aaaggtggtc cgctgccgtt cgcttgggac atcctgtccc cgcagttcca
     1561 gtacggttcc aaagcttacg ttaaacaccc ggctgacatc ccggactacc tgaaactgtc
     1621 cttcccggaa ggtttcaaat gggaacgtgt tatgaacttc gaagacggtg gtgttgttac
     1681 cgttacccag gactcctccc tgcaagacgg tgagttcatc tacaaagtta aactgcgtgg
     1741 taccaacttc ccgtccgacg gtccggttat gcagaaaaaa accatgggtt gggaagcttc
     1801 caccgaacgt atgtacccgg aagacggtgc tctgaaaggt gaaatcaaaa tgcgtctgaa
     1861 actgaaagac ggtggtcact acgacgctga agttaaaacc acctacatgg ctaaaaaacc
     1921 ggttcagctg ccgggtgctt acaaaaccga catcaaactg gacatcacct cccacaacga
     1981 agactacacc atcgttgaac agtacgaacg tgctgaaggt cgtcactcca ccggtgctta
     2041 aggatccaaa ctcgagtaag gatctccagg catcaaataa aacgaaaggc tcagtcgaaa
     2101 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta gagtcacact
     2161 ggctcacctt cgggtgggcc tttctgcgtt tatacctagg gtacgggttt tgctgcccgc
     2221 aaacgggctg ttctggtgtt gctagtttgt tatcagaatc gcagatccgg cttcagccgg
     2281 tttgccggct gaaagcgcta tttcttccag aattgccatg attttttccc cacgggaggc
     2341 gtcactggct cccgtgttgt cggcagcttt gattcgataa gcagcatcgc ctgtttcagg
     2401 ctgtctatgt gtgactgttg agctgtaaca agttgtctca ggtgttcaat ttcatgttct
     2461 agttgctttg ttttactggt ttcacctgtt ctattaggtg ttacatgctg ttcatctgtt
     2521 acattgtcga tctgttcatg gtgaacagct ttgaatgcac caaaaactcg taaaagctct
     2581 gatgtatcta tcttttttac accgttttca tctgtgcata tggacagttt tccctttgat
     2641 atgtaacggt gaacagttgt tctacttttg tttgttagtc ttgatgcttc actgatagat
     2701 acaagagcca taagaacctc agatccttcc gtatttagcc agtatgttct ctagtgtggt
     2761 tcgttgtttt tgcgtgagcc atgagaacga accattgaga tcatacttac tttgcatgtc
     2821 actcaaaaat tttgcctcaa aactggtgag ctgaattttt gcagttaaag catcgtgtag
     2881 tgtttttctt agtccgttat gtaggtagga atctgatgta atggttgttg gtattttgtc
     2941 accattcatt tttatctggt tgttctcaag ttcggttacg agatccattt gtctatctag
     3001 ttcaacttgg aaaatcaacg tatcagtcgg gcggcctcgc ttatcaacca ccaatttcat
     3061 attgctgtaa gtgtttaaat ctttacttat tggtttcaaa acccattggt taagcctttt
     3121 aaactcatgg tagttatttt caagcattaa catgaactta aattcatcaa ggctaatctc
     3181 tatatttgcc ttgtgagttt tcttttgtgt tagttctttt aataaccact cataaatcct
     3241 catagagtat ttgttttcaa aagacttaac atgttccaga ttatatttta tgaatttttt
     3301 taactggaaa agataaggca atatctcttc actaaaaact aattctaatt tttcgcttga
     3361 gaacttggca tagtttgtcc actggaaaat ctcaaagcct ttaaccaaag gattcctgat
     3421 ttccacagtt ctcgtcatca gctctctggt tgctttagct aatacaccat aagcattttc
     3481 cctactgatg ttcatcatct gagcgtattg gttataagtg aacgataccg tccgttcttt
     3541 ccttgtaggg ttttcaatcg tggggttgag tagtgccaca cagcataaaa ttagcttggt
     3601 ttcatgctcc gttaagtcat agcgactaat cgctagttca tttgctttga aaacaactaa
     3661 ttcagacata catctcaatt ggtctaggtg attttaatca ctataccaat tgagatgggc
     3721 tagtcaatga taattactag tccttttccc gggtgatctg ggtatctgta aattctgcta
     3781 gacctttgct ggaaaacttg taaattctgc tagaccctct gtaaattccg ctagaccttt
     3841 gtgtgttttt tttgtttata ttcaagtggt tataatttat agaataaaga aagaataaaa
     3901 aaagataaaa agaatagatc ccagccctgt gtataactca ctactttagt cagttccgca
     3961 gtattacaaa aggatgtcgc aaacgctgtt tgctcctcta caaaacagac cttaaaaccc
     4021 taaaggctta agtagcaccc tcgcaagctc gggcaaatcg ctgaatattc cttttgtctc
     4081 cgaccatcag gcacctgagt cgctgtcttt ttcgtgacat tcagttcgct gcgctcacgg
     4141 ctctggcagt gaatgggggt aaatggcact acaggcgcct tttatggatt catgcaagga
     4201 aactacccat aatacaagaa aagcccgtca cgggcttctc agggcgtttt atggcgggtc
     4261 tgctatgtgg tgctatctga ctttttgctg ttcagcagtt cctgccctct gattttcca
//
