LOCUS       sequence-345642-        3853 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes mScarlet fluorescent protein. To be used in cell-free 
            system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3853)
  AUTHORS   Guzman-Chavez F, Arce A, Adhikari A, Vadhin S, Pedroza-Garcia JA, 
            Gandini C, Ajioka JW, Molloy J, Sanchez-Nieto S, Varner JD, Federici
            F, Haseloff J
  TITLE     Constructing Cell-Free Expression Systems for Low-Cost Access.
  JOURNAL   ACS Synth Biol. 2022 Mar 18;11(3):1114-1128. doi: 
            10.1021/acssynbio.1c00342. Epub 2022 Mar 8.
  PUBMED    35259873
REFERENCE   2  (bases 1 to 3853)
  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: 
            "10.1021/acssynbio.1c00342"; journalName: "ACS Synth Biol"; date: 
            "2022-03-18- 18"; volume: "11"; issue: "3"; pages: "1114-1128"
COMMENT     Sequence Label: pT7-RiboJ-mScarlet-T7
FEATURES             Location/Qualifiers
     source          1..3853
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(48..636)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(128..147)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(807..1622)
                     /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="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1530..1549
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1623..1725)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     1793..1811
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1849..1871)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1971..1990
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2184..2206
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2198..2215
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2199..2215
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2401..2420
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2401..2419
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        2423..2472
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             2523..2545
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2553..3248
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(2686..2704)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2954..2973
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3268..3286)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3282..3329
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3499..3515)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3499..3515)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3512..3534)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3523..3539
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3547..3553,3554..3571,3572..3577))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3592..3613
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3730..3747)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
       61 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      121 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
      181 accctgtcgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
      241 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
      301 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
      361 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
      421 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
      481 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
      541 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
      601 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
      661 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
      721 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
      781 atatatgagt aaacttggtc tgacagttag aaaaactcat cgagcatcaa gtgaaactgc
      841 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
      901 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
      961 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     1021 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaagag tttatgcatt
     1081 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcacca
     1141 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgccgtta
     1201 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     1261 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttccctggg
     1321 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     1381 agaggcataa attccgtcag ccagtttagc ctgaccatct catctgtaac atcattggca
     1441 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     1501 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     1561 gcatccatgt tggaatttaa tcgcggcttc aagcaagacg tttcccgttg aatatggctc
     1621 attttagctt ccttagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat
     1681 cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcacacat ttccccgaaa
     1741 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     1801 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     1861 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     1921 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     1981 gcagattgta ctgagagtgc accaaatgcg gtgtgaaata ccgcacagat gcgtaaggag
     2041 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     2101 ggtgcgggcc tcatcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     2161 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgcaac
     2221 gcgatgacga tggatagcga ttcatcgatg agctgacccg atcgccgccg ccggagggtt
     2281 gcgtttgaga cgggcgacag atcttaagca ttactcgcat ccattctcag gctgtctcgt
     2341 ctcgtctccg gaagacatgc ttaggagcct gcattaggat cgatctcgat cccgcgaaat
     2401 taatacgact cactataggg agctgtcacc ggatgtgctt tccggtctga tgagtccgtg
     2461 aggacgaaac agcctctaca aataattttg tttaatacta gacagaaaca gaggagatat
     2521 gctttgttta actttaagaa ggagatatac aaatggtgag caagggcgag gcagtgatca
     2581 aggagttcat gcggttcaag gtgcacatgg agggctccat gaacggccac gagttcgaga
     2641 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
     2701 ccaagggtgg ccccctgccc ttctcctggg acatcctgtc ccctcagttc atgtacggct
     2761 ccagggcctt catcaagcac cccgccgaca tccccgacta ctataagcag tccttccccg
     2821 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgccgtg accgtgaccc
     2881 aggacacctc cctggaggac ggcaccctga tctacaaggt gaagctccgc ggcaccaact
     2941 tccctcctga cggccccgta atgcagaaga agacaatggg ctgggaagcg tccaccgagc
     3001 ggttgtaccc cgaggacggc gtgctgaagg gcgacattaa gatggccctg cgcctgaagg
     3061 acggcggccg ctacctggcg gacttcaaga ccacctacaa ggccaagaag cccgtgcaga
     3121 tgcccggcgc ctacaacgtc gaccgcaagt tggacatcac ctcccacaac gaggactaca
     3181 ccgtggtgga acagtacgaa cgctccgagg gccgccactc caccggcggc atggacgagc
     3241 tgtacaagta agcttaggtc gcctaggccg ctgagcaata actagcataa ccccttgggg
     3301 cctctaaacg ggtcttgagg ggttttttgc tgaaaacctc gctttgtcat gtcttcaggc
     3361 tgggagttcg tagacggaaa caaacgcaga atccaagccg cgcgcggcca ggatacatag
     3421 attaccacaa ctccgagccc ttccacccgc atcagttctg gaccagcgag ctgtgctgcg
     3481 actcgtggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     3541 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     3601 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     3661 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     3721 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     3781 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3841 catgtgagca aaa
//
