LOCUS       sequence-255886-        2588 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a fluorescent reporter for the quantification of cell-free
            protein expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2588)
  AUTHORS   Cole SD, Beabout K, Turner KB, Smith ZK, Funk VL, Harbaugh SV, Liem 
            AT, Roth PA, Geier BA, Emanuel PA, Walper SA, Chavez JL, Lux MW
  TITLE     Quantification of Interlaboratory Cell-Free Protein Synthesis 
            Variability.
  JOURNAL   ACS Synth Biol. 2019 Sep 20;8(9):2080-2091. doi: 
            10.1021/acssynbio.9b00178. Epub 2019 Sep 10.
  PUBMED    31386355
REFERENCE   2  (bases 1 to 2588)
  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.9b00178"; journalName: "ACS Synth Biol"; date: 
            "2019-09-20- 20"; volume: "8"; issue: "9"; pages: "2080-2091"
COMMENT     Sequence Label: pY71-PT7-RiboJ-sfGFP-MGapt
FEATURES             Location/Qualifiers
     source          1..2588
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      9..597
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     498..517
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     620..639
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        620..638
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        642..691
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             722..733
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             743..760
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             761..781
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1421..1468
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             1469..1492
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(1660..2475)
                     /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="MSHIQRETSCSKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2383..2402
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2467..2486
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
       61 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
      121 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
      181 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
      241 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
      301 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
      361 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
      421 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
      481 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
      541 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
      601 gccagatctc gatcccgcgt aatacgactc actataggga gctgtcaccg gatgtgcttt
      661 ccggtctgat gagtccgtga ggacgaaaca gcctctacaa ataattttgt ttaatctaga
      721 gaaagaggag aaatactaga tgcatcacca tcaccatcat gagaatttat acttccagag
      781 tagcaaaggt gaagaactgt ttaccggcgt tgtgccgatt ctggtggaac tggatggcga
      841 tgtgaacggt cacaaattca gcgtgcgtgg tgaaggtgaa ggcgatgcca cgattggcaa
      901 actgacgctg aaatttatct gcaccaccgg caaactgccg gtgccgtggc cgacgctggt
      961 gaccaccctg acctatggcg ttcagtgttt tagtcgctat ccggatcaca tgaaacgtca
     1021 cgatttcttt aaatctgcaa tgccggaagg ctatgtgcag gaacgtacga ttagctttaa
     1081 agatgatggc aaatataaaa cgcgcgccgt tgtgaaattt gaaggcgata ccctggtgaa
     1141 ccgcattgaa ctgaaaggca cggattttaa agaagatggc aatatcctgg gccataaact
     1201 ggaatacaac tttaatagcc ataatgttta tattacggcg gataaacaga aaaatggcat
     1261 caaagcgaat tttaccgttc gccataacgt tgaagatggc agtgtgcagc tggcagatca
     1321 ttatcagcag aataccccga ttggtgatgg tccggtgctg ctgccggata atcattatct
     1381 gagcacgcag accgttctgt ctaaagatcc gaacgaaaaa cgggaccaca tggttctgca
     1441 cgaatatgtg aatgcggcag gtattacgtg gagccatccg cagttcgaaa aataataaac
     1501 aataactgaa tagggatccc gactggcgag agccaggtaa cgaatggatc cttcagccaa
     1561 aaaacttaag accgccggtc ttgtccacta ccttgcagta atgcggtgga caggatcggc
     1621 ggttttcttt tctcttctca agctgaaagc caattctgat tagaaaaact catcgagcat
     1681 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     1741 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     1801 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     1861 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     1921 aagcttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     1981 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     2041 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     2101 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     2161 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     2221 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     2281 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     2341 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     2401 cccatataaa tcagcatcca tgttggaatt taatcgcggc ttcgagcaag acgtttcccg
     2461 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     2521 tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag acacaacgtg
     2581 agatcaaa
//
