LOCUS       sequence-381463-        5102 bp    DNA     circular SYN 14-APR-2025
DEFINITION  RFP GFP fusion protein expressed under a constitutive promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5102)
  AUTHORS   Wu WY, Mohanraju P, Liao C, Adiego-Perez B, Creutzburg SCA, Makarova
            KS, Keessen K, Lindeboom TA, Khan TS, Prinsen S, Joosten R, Yan WX, 
            Migur A, Laffeber C, Scott DA, Lebbink JHG, Koonin EV, Beisel CL, 
            van der Oost J
  TITLE     The miniature CRISPR-Cas12m effector binds DNA to block 
            transcription.
  JOURNAL   Mol Cell. 2022 Dec 1;82(23):4487-4502.e7. doi: 
            10.1016/j.molcel.2022.11.003. Epub 2022 Nov 24.
  PUBMED    36427491
REFERENCE   2  (bases 1 to 5102)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2022.11.003"; journalName: "Mol Cell"; date: 
            "2022-12-1- 1"; volume: "82"; issue: "23"; pages: "4487-4502.e7"
COMMENT     Sequence Label: pTarget-RFP-GFP operon
FEATURES             Location/Qualifiers
     source          1..5102
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     531..550
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     terminator      755..826
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             970..1647
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             1697..2410
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1727..1755)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2350..2370
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2451..2537
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3075..4025)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      4073..4295
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(4787..4881)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(4912..5102,1..604))
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5023..5042)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct
       61 cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat
      121 gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct
      181 cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc
      241 gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga
      301 gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt
      361 cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt
      421 cctgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct
      481 gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat
      541 agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa
      601 tcatgcgaaa cgatcctcat cctgtctctt gatcagatct tgatcccctg cgccatcaga
      661 tccttggcgg caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg
      721 gcgccccagc tggcaattcc gacgtcccag gcatcaaata aaacgaaagg ctcagtcgaa
      781 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcaggc cctttcgtct
      841 tccttagtcg agtgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
      901 ttcaggggga gaccacaacg gtttccctct agaaataatt ttgtttaact ataagaagga
      961 gatatacata tggcttcctc cgaagacgtt atcaaagagt tcatgcgttt caaagttcgt
     1021 atggaaggtt ccgttaacgg tcacgagttc gaaatcgaag gtgaaggtga aggtcgtccg
     1081 tacgaaggta cacagaccgc taaactgaaa gttaccaaag gtggcccgct gccgttcgct
     1141 tgggacatcc tgtccccgca gttccagtac ggttccaaag cttacgttaa acacccggct
     1201 gacatcccgg actacctgaa actgtccttc ccggaaggtt tcaaatggga acgtgttatg
     1261 aacttcgaag acggtggtgt tgttaccgtt acccaggact cctccctgca agacggtgag
     1321 ttcatctaca aagttaaact gcgtggtacc aacttcccgt ccgacggtcc ggttatgcag
     1381 aaaaaaacca tgggttggga agcttccacc gaacgtatgt acccggaaga cggtgctctg
     1441 aaaggtgaaa tcaaaatgcg tctgaaactg aaagacggtg gtcactacga cgctgaagtt
     1501 aaaaccacct acatggctaa aaaaccggtt cagctgccgg gtgcttacaa aaccgacatc
     1561 aaactggaca tcacctccca caacgaagac tacaccatcg ttgaacagta cgaacgtgct
     1621 gaaggtcgtc actccaccgg tgcttaaact agaaataatt ttgtttaacc tctagattta
     1681 agaaggagat atacatatga gtaaaggaga agaacttttc actggagttg tcccaattct
     1741 tgttgaatta gatggtgatg ttaatgggca caaattttct gtcagtggag agggtgaagg
     1801 tgatgcaaca tacggaaaac ttacccttaa atttatttgc actactggaa aactacctgt
     1861 tccatggcca acacttgtca ctactttcgc gtatggtctt caatgctttg cgagataccc
     1921 agatcatatg aaacagcatg actttttcaa gagtgccatg cccgaaggtt atgtacagga
     1981 aagaactata tttttcaaag atgacgggaa ctacaagaca cgtgctgaag tcaagtttga
     2041 aggtgatacc cttgttaata gaatcgagtt aaaaggtatt gattttaaag aagatggaaa
     2101 cattcttgga cacaaattgg aatacaacta taactcacac aatgtataca tcatggcaga
     2161 caaacaaaag aatggaatca aagttaactt caaaattaga cacaacattg aagatggaag
     2221 cgttcaacta gcagaccatt atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt
     2281 accagacaac cattacctgt ccacacaatc tgccctttcg aaagatccca acgaaaagag
     2341 agaccacatg gtccttcttg agtttgtaac agctgctggg attacacatg gcatggatga
     2401 actatacaaa taaatgtcca gacctgcagg catgcaagct ctagaggcat caaataaaac
     2461 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     2521 tcctgagtag gacaaatccg ccgccctaga cctagggtac gggttttgct gcccgcaaac
     2581 gggctgttct ggtgttgcta gtttgttatc agaatcgcag atccggcttc agccggtttg
     2641 ccggctgaaa gcgctatttc ttccagaatt gccatgattt tttccccacg ggaggcgtca
     2701 ctggctcccg tgttgtcggc agctttgatt cgataagcag catcgcctgt ttcaggctgt
     2761 ctatgtgtga ctgttgagct gtaacaagtt gtctcaggtg ttcaatttca tgttctagtt
     2821 gctttgtttt actggtttca cctgttctat taggtgttac atgctgttca tctgttacat
     2881 tgtcgatctg ttcatggtga acagctttga atgcaccaaa aactcgtaaa agctctgatg
     2941 tatctatctt ttttacaccg ttttcatctg tgcatatgga cagttttccc tttgatatgt
     3001 aacggtgaac agttgttcta cttttgtttg ttagtcttga tgcttcactg atagatacaa
     3061 gagccataag aacctcagat ccttccgtat ttagccagta tgttctctag tgtggttcgt
     3121 tgtttttgcg tgagccatga gaacgaacca ttgagatcat acttactttg catgtcactc
     3181 aaaaattttg cctcaaaact ggtgagctga atttttgcag ttaaagcatc gtgtagtgtt
     3241 tttcttagtc cgttatgtag gtaggaatct gatgtaatgg ttgttggtat tttgtcacca
     3301 ttcattttta tctggttgtt ctcaagttcg gttacgagat ccatttgtct atctagttca
     3361 acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat caaccaccaa tttcatattg
     3421 ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc attggttaag ccttttaaac
     3481 tcatggtagt tattttcaag cattaacatg aacttaaatt catcaaggct aatctctata
     3541 tttgccttgt gagttttctt ttgtgttagt tcttttaata accactcata aatcctcata
     3601 gagtatttgt tttcaaaaga cttaacatgt tccagattat attttatgaa tttttttaac
     3661 tggaaaagat aaggcaatat ctcttcacta aaaactaatt ctaatttttc gcttgagaac
     3721 ttggcatagt ttgtccactg gaaaatctca aagcctttaa ccaaaggatt cctgatttcc
     3781 acagttctcg tcatcagctc tctggttgct ttagctaata caccataagc attttcccta
     3841 ctgatgttca tcatctgagc gtattggtta taagtgaacg ataccgtccg ttctttcctt
     3901 gtagggtttt caatcgtggg gttgagtagt gccacacagc ataaaattag cttggtttca
     3961 tgctccgtta agtcatagcg actaatcgct agttcatttg ctttgaaaac aactaattca
     4021 gacatacatc tcaattggtc taggtgattt taatcactat accaattgag atgggctagt
     4081 caatgataat tactagtcct tttcctttga gttgtgggta tctgtaaatt ctgctagacc
     4141 tttgctggaa aacttgtaaa ttctgctaga ccctctgtaa attccgctag acctttgtgt
     4201 gttttttttg tttatattca agtggttata atttatagaa taaagaaaga ataaaaaaag
     4261 ataaaaagaa tagatcccag ccctgtgtat aactcactac tttagtcagt tccgcagtat
     4321 tacaaaagga tgtcgcaaac gctgtttgct cctctacaaa acagacctta aaaccctaaa
     4381 ggcttaagta gcaccctcgc aagctcgggc aaatcgctga atattccttt tgtctccgac
     4441 catcaggcac ctgagtcgct gtctttttcg tgacattcag ttcgctgcgc tcacggctct
     4501 ggcagtgaat gggggtaaat ggcactacag gcgcctttta tggattcatg caaggaaact
     4561 acccataata caagaaaagc ccgtcacggg cttctcaggg cgttttatgg cgggtctgct
     4621 atgtggtgct atctgacttt ttgctgttca gcagttcctg ccctctgatt ttccagtctg
     4681 accacttcgg attatcccgt gacaggtcat tcagactggc taatgcaccc agtaaggcag
     4741 cggtatcatc aacaggctta cccgtcttac tgtccctagt gcttggattc tcaccaataa
     4801 aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta
     4861 ctggatctat caacaggagt ccaagcgagc tctcgaaccc cagagtcccg ctcagaagaa
     4921 ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag
     4981 cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa
     5041 cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa
     5101 gc
//
