LOCUS       sequence-336158-        4591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes RRvT 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 4591)
  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 4591)
  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: pFGC-T7RibJ-RRvT
FEATURES             Location/Qualifiers
     source          1..4591
                     /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)"
     primer_bind     complement(866..884)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2186..2204)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2200..2247
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2417..2433)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2417..2433)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2430..2452)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2441..2457
                     /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(2465..2471,2472..2489,2490..2495))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2510..2531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2648..2665)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2819..3407)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2899..2918)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3578..4393)
                     /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     4301..4320
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4394..4496)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     4564..4582
                     /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 gaggtgatca aggaattcat gaggttcaag
      781 gtgaggatgg agggctccat gaatggacat gagtttgaaa ttgaaggaga gggagaggga
      841 cgcccttatg aaggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc
      901 ttcgcctggg acattctgtc ccctcagttt atgtatggat ctaaggctta tgtcaaacat
      961 cctgctgata ttcccgacta caagaagctg agcttccccg agggcttcaa gtgggagagg
     1021 gttatgaact tcgaagatgg aggactggtc acagtcacac aggattcctc cctgcaggat
     1081 ggaacactga tttacaatgt gaagatgagg ggcaccaact ttccacccga cggccccgtg
     1141 atgcaaaaga aaacaatggg atgggaggct tccacagaac gcctgtatcc tcgtgatgga
     1201 gtcctgaaag gagagatcca ccaggccctg aagctgaagg acggcggcca ctacctggtg
     1261 gagtttaaga ccatttatat ggctaaaaaa cctgtccaac tgcctggata ttattatgtc
     1321 gatacaaaac tggacatcac cagccacaac gaggactaca ccatcgtgga gcagtacgag
     1381 aggagcgagg gccgccatca tctgttcctc tatggaatgg atgaactcta taaaggcagc
     1441 accggcagcg gcagctccgg ccccatggtt tccaaaggag aagaagccat taaagagttt
     1501 atgcgcttca aagtcagcat ggaaggcagc atgaacggcc acgagttcga gatcgagggc
     1561 gagggcgagg gcaggcccta cgagggaaca cagacagcta aactgaaagt cacaaaagga
     1621 ggacctctgc ctttcgcttg ggatatcctg agcccccagt tcatgtacgg cagcaaggcc
     1681 tacgtgaagc accccgccga catccctgat tataaaaaac tgtcctttcc tgaaggattc
     1741 agatgggaac gcgtcatgaa tttcgaggac ggcggcctgg tgaccgtgac ccaggacagc
     1801 agcatccagg acggcaccct gatctataaa gtcaaagtgc gcggaacaaa tttccctcct
     1861 gatggacctg tcatgcagaa aaaaaccatg ggctgggaag ccagcaccga gaggctgtac
     1921 cccagggacg gcgtgctgaa gggcgaaatt catcaggctc tgaaactgaa agatggagga
     1981 cattatctgg tcgaattcaa aacaatctac atggccaaga agcccgtgca gctccccggc
     2041 tactactacg tggacaccaa gctggatatt acatcccata atgaagatta tacagttgtc
     2101 gaacagtatg aacgctccga aggaaggcac cacctctttc tgtacggcat ggacgagctg
     2161 tacaagtaag cttaggtcgc ctaggccgct gagcaataac tagcataacc ccttggggcc
     2221 tctaaacggg tcttgagggg ttttttgctg aaaacctcgc tttgtcatgt cttcaggctg
     2281 ggagttcgta gacggaaaca aacgcagaat ccaagccgcg cgcggccagg atacatagat
     2341 taccacaact ccgagccctt ccacccgcat cagttctgga ccagcgagct gtgctgcgac
     2401 tcgtggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     2461 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2521 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     2581 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     2641 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     2701 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2761 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2821 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2881 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2941 ctcctgttcc gaccctgtcg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3001 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3061 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3121 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3181 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3241 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3301 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3361 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3421 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3481 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3541 caatctaaag tatatatgag taaacttggt ctgacagtta gaaaaactca tcgagcatca
     3601 agtgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     3661 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     3721 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     3781 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaga
     3841 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     3901 cactcgcacc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     3961 gatcgccgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     4021 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     4081 ttttccctgg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     4141 tgatggtcgg aagaggcata aattccgtca gccagtttag cctgaccatc tcatctgtaa
     4201 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     4261 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     4321 catataaatc agcatccatg ttggaattta atcgcggctt caagcaagac gtttcccgtt
     4381 gaatatggct cattttagct tccttagctc ctgaaaatct cgataactca aaaaatacgc
     4441 ccggtagtga tcttatttca ttatggtgaa agttggaacc tcttacgtgc cgatcacaca
     4501 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     4561 aaaaataggc gtatcacgag gccctttcgt c
//
