LOCUS       sequence-336159-        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"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     581..600
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        581..599
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        603..652
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             703..725
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             733..1428
                     /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(866..884)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1134..1153
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1448..1466)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1462..1509
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1679..1695)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1679..1695)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1692..1714)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1703..1719
                     /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(1727..1733,1734..1751,1752..1757))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1772..1793
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1910..1927)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2081..2669)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2161..2180)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2840..3655)
                     /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     3563..3582
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3656..3758)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     3826..3844
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accaaatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcatcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgcaac gcgatgacga tggatagcga
      421 ttcatcgatg agctgacccg atcgccgccg ccggagggtt gcgtttgaga cgggcgacag
      481 atcttaagca ttactcgcat ccattctcag gctgtctcgt ctcgtctccg gaagacatgc
      541 ttaggagcct gcattaggat cgatctcgat cccgcgaaat taatacgact cactataggg
      601 agctgtcacc ggatgtgctt tccggtctga tgagtccgtg aggacgaaac agcctctaca
      661 aataattttg tttaatacta gacagaaaca gaggagatat gctttgttta actttaagaa
      721 ggagatatac aaatggtgag caagggcgag gcagtgatca aggagttcat gcggttcaag
      781 gtgcacatgg agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc
      841 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
      901 ttctcctggg acatcctgtc ccctcagttc atgtacggct ccagggcctt catcaagcac
      961 cccgccgaca tccccgacta ctataagcag tccttccccg agggcttcaa gtgggagcgc
     1021 gtgatgaact tcgaggacgg cggcgccgtg accgtgaccc aggacacctc cctggaggac
     1081 ggcaccctga tctacaaggt gaagctccgc ggcaccaact tccctcctga cggccccgta
     1141 atgcagaaga agacaatggg ctgggaagcg tccaccgagc ggttgtaccc cgaggacggc
     1201 gtgctgaagg gcgacattaa gatggccctg cgcctgaagg acggcggccg ctacctggcg
     1261 gacttcaaga ccacctacaa ggccaagaag cccgtgcaga tgcccggcgc ctacaacgtc
     1321 gaccgcaagt tggacatcac ctcccacaac gaggactaca ccgtggtgga acagtacgaa
     1381 cgctccgagg gccgccactc caccggcggc atggacgagc tgtacaagta agcttaggtc
     1441 gcctaggccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     1501 ggttttttgc tgaaaacctc gctttgtcat gtcttcaggc tgggagttcg tagacggaaa
     1561 caaacgcaga atccaagccg cgcgcggcca ggatacatag attaccacaa ctccgagccc
     1621 ttccacccgc atcagttctg gaccagcgag ctgtgctgcg actcgtggcg taatcatggt
     1681 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     1741 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     1801 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     1861 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     1921 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     1981 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2041 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2101 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2161 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgt
     2221 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2281 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     2341 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     2401 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     2461 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     2521 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     2581 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     2641 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     2701 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     2761 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     2821 agtaaacttg gtctgacagt tagaaaaact catcgagcat caagtgaaac tgcaatttat
     2881 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     2941 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     3001 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     3061 aatcaccatg agtgacgact gaatccggtg agaatggcaa gagtttatgc atttctttcc
     3121 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca ccaaccaaac
     3181 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgccg ttaaaaggac
     3241 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     3301 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttccct gggatcgcag
     3361 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     3421 taaattccgt cagccagttt agcctgacca tctcatctgt aacatcattg gcaacgctac
     3481 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     3541 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     3601 tgttggaatt taatcgcggc ttcaagcaag acgtttcccg ttgaatatgg ctcattttag
     3661 cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt
     3721 cattatggtg aaagttggaa cctcttacgt gccgatcaca catttccccg aaaagtgcca
     3781 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     3841 aggccctttc gtc
//
