LOCUS       sequence-234641-        6513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RFP and GFP reporter genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6513)
  AUTHORS   Dong C, Fontana J, Patel A, Carothers JM, Zalatan JG
  TITLE     Synthetic CRISPR-Cas gene activators for transcriptional 
            reprogramming in bacteria.
  JOURNAL   Nat Commun. 2018 Jun 27;9(1):2489. doi: 10.1038/s41467-018-04901-6.
  PUBMED    29950558
REFERENCE   2  (bases 1 to 6513)
  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: "Nat 
            Commun."; date: "2018-06-27"; volume: "9(1):2489. doi"; pages: " 
            10.1038/s41467-018-04901-6"
COMMENT     Sequence Label: pCD002
FEATURES             Location/Qualifiers
     source          1..6513
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             549..578
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     promoter        788..822
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     RBS             832..843
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(850..852,853..1566)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             850..1563
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(880..908)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1503..1523
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1599..1670
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1686..1713
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             1859..1870
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1877..2554
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      2587..2658
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2674..2701
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    complement(2797..2830)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(2975..2994)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2986..3801
                     /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             2986..3801
                     /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     complement(3059..3078)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     protein_bind    complement(3865..3898)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     terminator      4399..4645
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     rep_origin      4706..5094
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(join(5196..5987,5988..6056))
                     /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     5819..5838
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6057..6161)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      6251..6337
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6429..6456
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gaaagtaggt ttaacctgtt gcattaattg ctaaaagcta taactgttaa acacaataca
       61 gtgaaaagtt ttagactgaa ggctcacttt gcagagggaa gcgtatgcgc gcgatcggta
      121 aattgcctaa aggcgtgttg atactggaat ttatcggaat gatgctactg gcggtggcgc
      181 tgctgtcggt aagcgactcc ctgtcgctgc ctgagccatt ttctcggcca gaagtgcaga
      241 ttctgatgat ttttctcggt gttttgctca tgcttcccgc tgcggtggtg gttattcttc
      301 aggtggcaaa acgtcttgcc ccacagctga tgaaccgtcc accgcaatat tcacgttcag
      361 aaagagaaaa ggataatgac gccaaccatt gaacttattt gtggccatcg ctccattcgc
      421 catttcactg atgaacccat ttccgaagcg cagcgtgagg cgattattaa cagcgcccgt
      481 gcgacgtcca gttccagttt tttggcatgc ccgatcaacg tctcattttc gccagatatc
      541 aagcagagga gcaaaagctc atttctgaag aggacttgtt gcggaaacga cgagaacagt
      601 tgaaacacaa acttgaacag ctacggaact cttgtgcgta aggaaaagta aggaaaacga
      661 ttccttctaa cagaaatgtc ctgagcaatc acctatgaac tgtcgactcg agcctctatg
      721 gattatcacc ttggctgcag gccggatctt ccacaacacg cacggtgtta cattaggcat
      781 accggtcttg acagctagct cagtcctagg gattgtgcta gcgaattcat taaagaggag
      841 aaaggtacca tgagcaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa
      901 ttagatggtg atgttaatgg gcacaaattt tctgtccgtg gagagggtga aggtgatgct
      961 acaaacggaa aactcaccct taaatttatt tgcactactg gaaaactacc tgttccgtgg
     1021 ccaacacttg tcactactct gacctatggt gttcaatgct tttcccgtta tccggatcac
     1081 atgaaacggc atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaacgcact
     1141 atatctttca aagatgacgg gacctacaag acgcgtgctg aagtcaagtt tgaaggtgat
     1201 acccttgtta atcgtatcga gttaaagggt attgatttta aagaagatgg aaacattctt
     1261 ggacacaaac tcgagtacaa ctttaactca cacaatgtat acatcacggc agacaaacaa
     1321 aagaatggaa tcaaagctaa cttcaaaatt cgccacaacg ttgaagatgg ttccgttcaa
     1381 ctagcagacc attatcaaca aaatactcca attggcgatg gccctgtcct tttaccagac
     1441 aaccattacc tgtcgacaca atctgtcctt tcgaaagatc ccaacgaaaa gcgtgaccac
     1501 atggtccttc ttgagtttgt aactgctgct gggattacac atggcatgga tgagctctac
     1561 aaataaggat ccaaactcga gtaaggatct ccaggcatca aataaaacga aaggctcagt
     1621 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc
     1681 acactggctc accttcgggt gggcctttct gcgtttatac ctagggtacg ggttttgctg
     1741 ccctctagag gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     1801 tcagggtggt gaatttgaca gctagctcag tcctaggtat aatagatctg aattcattaa
     1861 agaggagaaa ggtaccatgg cgagtagcga agacgttatc aaagagttca tgcgtttcaa
     1921 agttcgtatg gaaggttccg ttaacggtca cgagttcgaa atcgaaggtg aaggtgaagg
     1981 tcgtccgtac gaaggtaccc agaccgctaa actgaaagtt accaaaggtg gtccgctgcc
     2041 gttcgcttgg gacatcctgt ccccgcagtt ccagtacggt tccaaagctt acgttaaaca
     2101 cccggctgac atcccggact acctgaaact gtccttcccg gaaggtttca aatgggaacg
     2161 tgttatgaac ttcgaagacg gtggtgttgt taccgttacc caggactcct ccctgcaaga
     2221 cggtgagttc atctacaaag ttaaactgcg tggtaccaac ttcccgtccg acggtccggt
     2281 tatgcagaaa aaaaccatgg gttgggaagc ttccaccgaa cgtatgtacc cggaagacgg
     2341 tgctctgaaa ggtgaaatca aaatgcgtct gaaactgaaa gacggtggtc actacgacgc
     2401 tgaagttaaa accacctaca tggctaaaaa accggttcag ctgccgggtg cttacaaaac
     2461 cgacatcaaa ctggacatca cctcccacaa cgaagactac accatcgttg aacagtacga
     2521 acgtgctgaa ggtcgtcact ccaccggtgc ttaaggatcc aaactcgagt aaggatctcc
     2581 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     2641 ttgtcggtga acgctctcta ctagagtcac actggctcac cttcgggtgg gcctttctgc
     2701 gtttatacct agggtacggg ttttgctgcc cgcaaacggg ctgttctggt gttgactagt
     2761 ggatgaactg tacaaatgag gatcctaaag atctgcgaag ttcctatact ttctagagaa
     2821 taggaacttc ggatctgctg atccttcaac tcagcaaaag ttcgatttat tcaacaaagc
     2881 cacgttgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga
     2941 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     3001 cgggaaacgt cttgctccag gccgcgatta aattccaaca tggatgctga tttatatggg
     3061 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg
     3121 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
     3181 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
     3241 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca
     3301 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
     3361 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
     3421 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
     3481 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataagctt
     3541 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
     3601 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
     3661 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
     3721 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
     3781 atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt
     3841 acgctacttg acgggaggat ctgcgaagtt cctatacttt ctagagaata ggaacttcgg
     3901 atcttggcgg aatattacct cacccgtggc agcaataatc gccgggatac ctggagcgat
     3961 catatccgcc gaacaatcat taaagaaagc cgcccattta ttctggatta tttgcacaaa
     4021 cagggttggg cgacgcgcta aaaccgccac gtcgatgtat gatacgcggg cttttgacca
     4081 ggtctgacag agaggtgcag ggtgaaaatt gccatattgt cccgggatgg aacgctctat
     4141 tcgtgtaagc ggctgcgtga agccgctata cagcgcggtc acctggttga aattcttgat
     4201 ccgctttctt gctacatgaa cataaatcct gcggcgtctt ctattcacta caaaggccgc
     4261 aagttacccc attttgacgc agtgatcccg cgtattggca ccgccattac cttttatggg
     4321 acggcggcac tgcgccagtt cgagatgctg gggagctatc cgctcactta tcactgatca
     4381 gtgaattaat ggcgatgacg catcctcacg ataatatccg ggtaggcgca atcactttcg
     4441 tctctactcc gttacaaagc gaggctgggt atttcccggc ctttctgtta tccgaaatcc
     4501 actgaaagca cagcggctgg ctgaggagat aaataataaa cgaggggctg tatgcacaaa
     4561 gcatcttctg ttgagttaag aacgagtatc gagatggcac atagccttgc tcaaattgga
     4621 atcaggtttg tgccaatacc agtagaaaca gacgaagaag ctagctttgc actggattgc
     4681 gaggctttgc catggctaat tcccatgtca gccgttaagt gttcctgtgt cactcaaaat
     4741 tgctttgaga ggctctaagg gcttctcagt gcgttacatc cctggcttgt tgtccacaac
     4801 cgttaaacct taaaagcttt aaaagcctta tatattcttt tttttcttat aaaacttaaa
     4861 accttagagg ctatttaagt tgctgattta tattaatttt attgttcaaa catgagagct
     4921 tagtacgtga aacatgagag cttagtacgt tagccatgag agcttagtac gttagccatg
     4981 agggtttagt tcgttaaaca tgagagctta gtacgttaaa catgagagct tagtacgtga
     5041 aacatgagag cttagtacgt actatcaaca ggttgaactg ctgatcttca gatcctctac
     5101 gccggacgca tcgtggccgg atcttgcggc cgcaaaaatt aaaaatgaag ttttaaatca
     5161 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5221 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5281 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5341 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5401 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5461 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5521 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5581 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5641 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5701 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5761 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5821 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5881 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5941 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6001 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6061 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6121 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6181 ccacctgcat cgatggcccc cgatggtagt gtggggtctc cccatgcgag agtagggaac
     6241 tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     6301 ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt
     6361 tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggcatca
     6421 aattaagcag aaggccatcc tgacggatgg cctttttgcg tggccagtgc caagcttgca
     6481 tgcagattgc agcattacac gtcttgagcg att
//
