LOCUS       sequence-449489-        5125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Prokaryotic expression of mRFP and GFP. Target sequences are 
            inserted in place of the RFP gene through AatII/KpnI restriction 
            ligation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5125)
  AUTHORS   van der Oost
  TITLE     van der Oost Lab Plasmids 2024
  JOURNAL   Unpublished
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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pTarget-entry
FEATURES             Location/Qualifiers
     source          1..5125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(26..820)
                     /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(137..156)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     747..766
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(987..1664)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     promoter        complement(join(1732..1738,1739..1754,1755..1760))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             1936..2649
                     /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(1966..1994)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2589..2609
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2690..2776
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3314..4264)
                     /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      4312..4534
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(5026..5120)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 gagctctcga accccagagt cccgctcaga agaactcgtc aagaaggcga tagaaggcga
       61 tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc
      121 cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca
      181 cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg
      241 gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga
      301 gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat
      361 cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt
      421 cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg
      481 atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca
      541 atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc
      601 ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg
      661 acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg
      721 catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag
      781 cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat cctcatcctg
      841 tctcttgatc agatcttgat cccctgcgcc atcagatcct tggcggcaag aaagccatcc
      901 agtttacttt gcagggcttc ccaaccttac cagagggcgc cccagctggc aattccgacg
      961 tccgttcatt aaaaattggc ggccgcttaa gcaccggtgg agtgacgacc ttcagcacgt
     1021 tcgtactgtt caacgatggt gtagtcttcg ttgtgggagg tgatgtccag tttgatgtcg
     1081 gttttgtaag cacccggcag ctgaaccggt tttttagcca tgtaggtggt tttaacttca
     1141 gcgtcgtagt gaccaccgtc tttcagtttc agacgcattt tgatttcacc tttcagagca
     1201 ccgtcttccg ggtacatacg ttcggtggaa gcttcccaac ccatggtttt tttctgcata
     1261 accggaccgt cggacgggaa gttggtacca cgcagtttaa ctttgtagat gaactcaccg
     1321 tcttgcaggg aggagtcctg ggtaacggta acaacaccac cgtcttcgaa gttcataaca
     1381 cgttcccatt tgaaaccttc cgggaaggac agtttcaggt agtccgggat gtcagccggg
     1441 tgtttaacgt aagctttgga accgtactgg aactgcgggg acaggatgtc ccaagcgaac
     1501 ggcagcgggc cacctttggt aactttcagt ttagcggtct gtgtaccttc gtacggacga
     1561 ccttcacctt caccttcgat ttcgaactcg tgaccgttaa cggaaccttc catacgaact
     1621 ttgaaacgca tgaactcttt gataacgtct tcggaggaag ccatatgtat atctccttct
     1681 tatagttaaa caaaattatt tctagaggga aaccgttgtg gtctccccac acattatacg
     1741 agccgatgat taattgtcaa cttgaaaaaa ccccgccgaa gcggggtacc cattaaccta
     1801 taaaaatagg cgtcgaggcc ctttcgtctt ccttagtcga gtgcaaaacc tttcgcggta
     1861 tggcatgata gcgcccggaa gagagtcaat tcaggggatc cggggatcct ctagatttaa
     1921 gaaggagata tacatatgag taaaggagaa gaacttttca ctggagttgt cccaattctt
     1981 gttgaattag atggtgatgt taatgggcac aaattttctg tcagtggaga gggtgaaggt
     2041 gatgcaacat acggaaaact tacccttaaa tttatttgca ctactggaaa actacctgtt
     2101 ccatggccaa cacttgtcac tactttcgcg tatggtcttc aatgctttgc gagataccca
     2161 gatcatatga aacagcatga ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa
     2221 agaactatat ttttcaaaga tgacgggaac tacaagacac gtgctgaagt caagtttgaa
     2281 ggtgataccc ttgttaatag aatcgagtta aaaggtattg attttaaaga agatggaaac
     2341 attcttggac acaaattgga atacaactat aactcacaca atgtatacat catggcagac
     2401 aaacaaaaga atggaatcaa agttaacttc aaaattagac acaacattga agatggaagc
     2461 gttcaactag cagaccatta tcaacaaaat actccaattg gcgatggccc tgtcctttta
     2521 ccagacaacc attacctgtc cacacaatct gccctttcga aagatcccaa cgaaaagaga
     2581 gaccacatgg tccttcttga gtttgtaaca gctgctggga ttacacatgg catggatgaa
     2641 ctatacaaat aaatgtccag acctgcaggc atgcaagctc tagaggcatc aaataaaacg
     2701 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
     2761 cctgagtagg acaaatccgc cgccctagac ctagggtacg ggttttgctg cccgcaaacg
     2821 ggctgttctg gtgttgctag tttgttatca gaatcgcaga tccggcttca gccggtttgc
     2881 cggctgaaag cgctatttct tccagaattg ccatgatttt ttccccacgg gaggcgtcac
     2941 tggctcccgt gttgtcggca gctttgattc gataagcagc atcgcctgtt tcaggctgtc
     3001 tatgtgtgac tgttgagctg taacaagttg tctcaggtgt tcaatttcat gttctagttg
     3061 ctttgtttta ctggtttcac ctgttctatt aggtgttaca tgctgttcat ctgttacatt
     3121 gtcgatctgt tcatggtgaa cagctttgaa tgcaccaaaa actcgtaaaa gctctgatgt
     3181 atctatcttt tttacaccgt tttcatctgt gcatatggac agttttccct ttgatatgta
     3241 acggtgaaca gttgttctac ttttgtttgt tagtcttgat gcttcactga tagatacaag
     3301 agccataaga acctcagatc cttccgtatt tagccagtat gttctctagt gtggttcgtt
     3361 gtttttgcgt gagccatgag aacgaaccat tgagatcata cttactttgc atgtcactca
     3421 aaaattttgc ctcaaaactg gtgagctgaa tttttgcagt taaagcatcg tgtagtgttt
     3481 ttcttagtcc gttatgtagg taggaatctg atgtaatggt tgttggtatt ttgtcaccat
     3541 tcatttttat ctggttgttc tcaagttcgg ttacgagatc catttgtcta tctagttcaa
     3601 cttggaaaat caacgtatca gtcgggcggc ctcgcttatc aaccaccaat ttcatattgc
     3661 tgtaagtgtt taaatcttta cttattggtt tcaaaaccca ttggttaagc cttttaaact
     3721 catggtagtt attttcaagc attaacatga acttaaattc atcaaggcta atctctatat
     3781 ttgccttgtg agttttcttt tgtgttagtt cttttaataa ccactcataa atcctcatag
     3841 agtatttgtt ttcaaaagac ttaacatgtt ccagattata ttttatgaat ttttttaact
     3901 ggaaaagata aggcaatatc tcttcactaa aaactaattc taatttttcg cttgagaact
     3961 tggcatagtt tgtccactgg aaaatctcaa agcctttaac caaaggattc ctgatttcca
     4021 cagttctcgt catcagctct ctggttgctt tagctaatac accataagca ttttccctac
     4081 tgatgttcat catctgagcg tattggttat aagtgaacga taccgtccgt tctttccttg
     4141 tagggttttc aatcgtgggg ttgagtagtg ccacacagca taaaattagc ttggtttcat
     4201 gctccgttaa gtcatagcga ctaatcgcta gttcatttgc tttgaaaaca actaattcag
     4261 acatacatct caattggtct aggtgatttt aatcactata ccaattgaga tgggctagtc
     4321 aatgataatt actagtcctt ttcctttgag ttgtgggtat ctgtaaattc tgctagacct
     4381 ttgctggaaa acttgtaaat tctgctagac cctctgtaaa ttccgctaga cctttgtgtg
     4441 ttttttttgt ttatattcaa gtggttataa tttatagaat aaagaaagaa taaaaaaaga
     4501 taaaaagaat agatcccagc cctgtgtata actcactact ttagtcagtt ccgcagtatt
     4561 acaaaaggat gtcgcaaacg ctgtttgctc ctctacaaaa cagaccttaa aaccctaaag
     4621 gcttaagtag caccctcgca agctcgggca aatcgctgaa tattcctttt gtctccgacc
     4681 atcaggcacc tgagtcgctg tctttttcgt gacattcagt tcgctgcgct cacggctctg
     4741 gcagtgaatg ggggtaaatg gcactacagg cgccttttat ggattcatgc aaggaaacta
     4801 cccataatac aagaaaagcc cgtcacgggc ttctcagggc gttttatggc gggtctgcta
     4861 tgtggtgcta tctgactttt tgctgttcag cagttcctgc cctctgattt tccagtctga
     4921 ccacttcgga ttatcccgtg acaggtcatt cagactggct aatgcaccca gtaaggcagc
     4981 ggtatcatca acaggcttac ccgtcttact gtccctagtg cttggattct caccaataaa
     5041 aaacgcccgg cggcaaccga gcgttctgaa caaatccaga tggagttctg aggtcattac
     5101 tggatctatc aacaggagtc caagc
//
