LOCUS       sequence-387959-        5125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RFP expressed under constitutive promoter Ptaq and GFP expressed 
            under constitutive promoter Placiq.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5125)
  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 5125)
  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: 
            "10.1016/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: Ptaq-RFP_PlacIq-GFP
FEATURES             Location/Qualifiers
     source          1..5125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      48..270
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(762..856)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(887..1681)
                     /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(998..1017)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1608..1627
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(1848..2525)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     promoter        complement(join(2593..2599,2600..2615,2616..2621))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             2797..3510
                     /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(2827..2855)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3450..3470
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3551..3637
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4175..5125)
                     /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 acatctcaat tggtctaggt gattttaatc actataccaa ttgagatggg ctagtcaatg
       61 ataattacta gtccttttcc tttgagttgt gggtatctgt aaattctgct agacctttgc
      121 tggaaaactt gtaaattctg ctagaccctc tgtaaattcc gctagacctt tgtgtgtttt
      181 ttttgtttat attcaagtgg ttataattta tagaataaag aaagaataaa aaaagataaa
      241 aagaatagat cccagccctg tgtataactc actactttag tcagttccgc agtattacaa
      301 aaggatgtcg caaacgctgt ttgctcctct acaaaacaga ccttaaaacc ctaaaggctt
      361 aagtagcacc ctcgcaagct cgggcaaatc gctgaatatt ccttttgtct ccgaccatca
      421 ggcacctgag tcgctgtctt tttcgtgaca ttcagttcgc tgcgctcacg gctctggcag
      481 tgaatggggg taaatggcac tacaggcgcc ttttatggat tcatgcaagg aaactaccca
      541 taatacaaga aaagcccgtc acgggcttct cagggcgttt tatggcgggt ctgctatgtg
      601 gtgctatctg actttttgct gttcagcagt tcctgccctc tgattttcca gtctgaccac
      661 ttcggattat cccgtgacag gtcattcaga ctggctaatg cacccagtaa ggcagcggta
      721 tcatcaacag gcttacccgt cttactgtcc ctagtgcttg gattctcacc aataaaaaac
      781 gcccggcggc aaccgagcgt tctgaacaaa tccagatgga gttctgaggt cattactgga
      841 tctatcaaca ggagtccaag cgagctctcg aaccccagag tcccgctcag aagaactcgt
      901 caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg taaagcacga
      961 ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta gccaacgcta
     1021 tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca gaaaagcggc
     1081 cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg agatcctcgc
     1141 cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc ccctgatgct
     1201 cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt gctcgctcga
     1261 tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc
     1321 gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat gacaggagat
     1381 cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg acaacgtcga
     1441 gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct
     1501 gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg
     1561 ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc cagtcatagc
     1621 cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct tgttcaatca
     1681 tgcgaaacga tcctcatcct gtctcttgat cagatcttga tcccctgcgc catcagatcc
     1741 ttggcggcaa gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg
     1801 ccccagctgg caattccgac gtccgttcat taaaaattgg cggccgctta agcaccggtg
     1861 gagtgacgac cttcagcacg ttcgtactgt tcaacgatgg tgtagtcttc gttgtgggag
     1921 gtgatgtcca gtttgatgtc ggttttgtaa gcacccggca gctgaaccgg ttttttagcc
     1981 atgtaggtgg ttttaacttc agcgtcgtag tgaccaccgt ctttcagttt cagacgcatt
     2041 ttgatttcac ctttcagagc accgtcttcc gggtacatac gttcggtgga agcttcccaa
     2101 cccatggttt ttttctgcat aaccggaccg tcggacggga agttggtacc acgcagttta
     2161 actttgtaga tgaactcacc gtcttgcagg gaggagtcct gggtaacggt aacaacacca
     2221 ccgtcttcga agttcataac acgttcccat ttgaaacctt ccgggaagga cagtttcagg
     2281 tagtccggga tgtcagccgg gtgtttaacg taagctttgg aaccgtactg gaactgcggg
     2341 gacaggatgt cccaagcgaa cggcagcggg ccacctttgg taactttcag tttagcggtc
     2401 tgtgtacctt cgtacggacg accttcacct tcaccttcga tttcgaactc gtgaccgtta
     2461 acggaacctt ccatacgaac tttgaaacgc atgaactctt tgataacgtc ttcggaggaa
     2521 gccatatgta tatctccttc ttatagttaa acaaaattat ttctagaggg aaaccgttgt
     2581 ggtctcccca cacattatac gagccgatga ttaattgtca acttgaaaaa accccgccga
     2641 agcggggtac ccattaacct ataaaaatag gcgtcgaggc cctttcgtct tccttagtcg
     2701 agtgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcaggggat
     2761 ccggggatcc tctagattta agaaggagat atacatatga gtaaaggaga agaacttttc
     2821 actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca caaattttct
     2881 gtcagtggag agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc
     2941 actactggaa aactacctgt tccatggcca acacttgtca ctactttcgc gtatggtctt
     3001 caatgctttg cgagataccc agatcatatg aaacagcatg actttttcaa gagtgccatg
     3061 cccgaaggtt atgtacagga aagaactata tttttcaaag atgacgggaa ctacaagaca
     3121 cgtgctgaag tcaagtttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt
     3181 gattttaaag aagatggaaa cattcttgga cacaaattgg aatacaacta taactcacac
     3241 aatgtataca tcatggcaga caaacaaaag aatggaatca aagttaactt caaaattaga
     3301 cacaacattg aagatggaag cgttcaacta gcagaccatt atcaacaaaa tactccaatt
     3361 ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc tgccctttcg
     3421 aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac agctgctggg
     3481 attacacatg gcatggatga actatacaaa taaatgtcca gacctgcagg catgcaagct
     3541 ctagaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     3601 ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgccctaga cctagggtac
     3661 gggttttgct gcccgcaaac gggctgttct ggtgttgcta gtttgttatc agaatcgcag
     3721 atccggcttc agccggtttg ccggctgaaa gcgctatttc ttccagaatt gccatgattt
     3781 tttccccacg ggaggcgtca ctggctcccg tgttgtcggc agctttgatt cgataagcag
     3841 catcgcctgt ttcaggctgt ctatgtgtga ctgttgagct gtaacaagtt gtctcaggtg
     3901 ttcaatttca tgttctagtt gctttgtttt actggtttca cctgttctat taggtgttac
     3961 atgctgttca tctgttacat tgtcgatctg ttcatggtga acagctttga atgcaccaaa
     4021 aactcgtaaa agctctgatg tatctatctt ttttacaccg ttttcatctg tgcatatgga
     4081 cagttttccc tttgatatgt aacggtgaac agttgttcta cttttgtttg ttagtcttga
     4141 tgcttcactg atagatacaa gagccataag aacctcagat ccttccgtat ttagccagta
     4201 tgttctctag tgtggttcgt tgtttttgcg tgagccatga gaacgaacca ttgagatcat
     4261 acttactttg catgtcactc aaaaattttg cctcaaaact ggtgagctga atttttgcag
     4321 ttaaagcatc gtgtagtgtt tttcttagtc cgttatgtag gtaggaatct gatgtaatgg
     4381 ttgttggtat tttgtcacca ttcattttta tctggttgtt ctcaagttcg gttacgagat
     4441 ccatttgtct atctagttca acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat
     4501 caaccaccaa tttcatattg ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc
     4561 attggttaag ccttttaaac tcatggtagt tattttcaag cattaacatg aacttaaatt
     4621 catcaaggct aatctctata tttgccttgt gagttttctt ttgtgttagt tcttttaata
     4681 accactcata aatcctcata gagtatttgt tttcaaaaga cttaacatgt tccagattat
     4741 attttatgaa tttttttaac tggaaaagat aaggcaatat ctcttcacta aaaactaatt
     4801 ctaatttttc gcttgagaac ttggcatagt ttgtccactg gaaaatctca aagcctttaa
     4861 ccaaaggatt cctgatttcc acagttctcg tcatcagctc tctggttgct ttagctaata
     4921 caccataagc attttcccta ctgatgttca tcatctgagc gtattggtta taagtgaacg
     4981 ataccgtccg ttctttcctt gtagggtttt caatcgtggg gttgagtagt gccacacagc
     5041 ataaaattag cttggtttca tgctccgtta agtcatagcg actaatcgct agttcatttg
     5101 ctttgaaaac aactaattca gacat
//
