LOCUS       sequence-374367-        4850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Standard for cell-free expression benchmarking..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4850)
  AUTHORS   Gagoski D, Polinkovsky ME, Mureev S, Kunert A, Johnston W, Gambin Y,
            Alexandrov K
  TITLE     Performance benchmarking of four cell-free protein expression 
            systems.
  JOURNAL   Biotechnol Bioeng. 2015 Aug 24. doi: 10.1002/bit.25814.
  PUBMED    26301602
REFERENCE   2  (bases 1 to 4850)
  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: "Biotechnol 
            Bioeng. 2015 Aug 24. doi: 10.1002/bit.25814."
COMMENT     Sequence Label: pCellFree_G03 RUVBL1
FEATURES             Location/Qualifiers
     source          1..4850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             183..206
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             213..923
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             213..923
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(252..273)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(513..532)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     860..881
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             924..947
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    948..972
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2341..2365)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      2589..2635
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2713..2729)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2713..2729)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2726..2748)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2737..2753
                     /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(2761..2767,2768..2785,2786..2791))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2806..2827
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2944..2961)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3115..3703)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3195..3214)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3874..4665,4666..4734))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERTRQIAEIGAS
                     LIKHW"
     primer_bind     4497..4516
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4735..4839)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaaaagtgcc acctgacgtc taatacgact cactataggg acatcttaag tttattttat
       61 tttattttat tttattttat tttattttat tttattttat tttattttat ttaaccatga
      121 cagtaatgta taaagtctgt aaagacatta aacacgtaag tgaaaccatg ggcacctctg
      181 gtcaccacca tcaccaccat caccacggca caagcaaggg cgaggagctg ttcaccgggg
      241 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
      301 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
      361 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
      421 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
      481 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
      541 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
      601 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
      661 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
      721 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
      781 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
      841 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
      901 tcggcatgga cgagctatac aagctggagg tgctcttcca gggtccgaca agtttgtaca
      961 aaaaagttgg cttgaagatt gaggaggtga agagcactac gaagacgcag cgcatcgcct
     1021 cccacagcca cgtgaaaggg ctggggctgg acgagagcgg cttggccaag caggcggcct
     1081 cagggcttgt gggccaggag aacgcgcgag aggcatgtgg cgtcatagta gaattaatca
     1141 aaagcaagaa aatggctgga agagctgtct tgttggcagg acctcctgga actggcaaga
     1201 cagctctggc tctggctatt gctcaggagc tgggtagtaa ggtccccttc tgcccaatgg
     1261 tggggagtga agtttactca actgagatca agaagacaga ggtgctgatg gagaacttcc
     1321 gcagggccat tgggctgcga ataaaggaga ccaaggaagt ttatgaaggt gaagtcacag
     1381 agctaactcc gtgtgagaca gagaatccca tgggaggata tggcaaaacc attagccatg
     1441 tgatcatagg actcaaaaca gccaaaggaa ccaaacagtt gaaactggac cccagcattt
     1501 ttgaaagttt gcagaaagag cgagtagaag ctggagatgt gatttacatt gaagccaaca
     1561 gtggggccgt gaagaggcag ggcaggtgtg atacctatgc cacagaattc gaccttgaag
     1621 ctgaagagta tgtccccttg ccaaaagggg atgtgcacaa aaagaaagaa atcatccaag
     1681 atgtgacctt gcatgacttg gatgtggcta atgcgcggcc ccagggggga caagatatcc
     1741 tgtccatgat gggccagcta atgaagccaa agaagacaga aatcacagac aaacttcgag
     1801 gggagattaa taaggtggtg aacaagtaca tcgaccaggg cattgctgag ctggtcccgg
     1861 gtgtgctgtt tgttgatgag gtccacatgc tggacattga gtgcttcacc tacctgcacc
     1921 gcgccctgga gtcttctatc gctcccatcg tcatctttgc atccaaccga ggcaactgtg
     1981 tcatcagagg cactgaggac atcacatccc ctcacggcat ccctcttgac cttctggacc
     2041 gagtgatgat aatccggacc atgctgtata ctccacagga aatgaaacag atcattaaaa
     2101 tccgtgccca gacggaagga atcaacatca gtgaggaggc actgaaccac ctgggggaga
     2161 ttggcaccaa gaccacactg aggtactcag tgcagctgct gaccccggcc aacttgcttg
     2221 ctaaaatcaa cgggaaggac agcattgaga aagagcatgt cgaagagatc agtgaacttt
     2281 tctatgatgc caagtcctcc gccaaaatcc tggctgacca gcaggataag tacatgaagt
     2341 gcccaacttt cttgtacaaa gtggtgtaag cggccgccct cctcctcctt tcttgttcct
     2401 ttcacgtcgc cttctcggtt gtagctggca gacgacgagt cttactttta cgtgtacttc
     2461 tctatagatg atgtatgatc tctctgcatg cgtgttcgtg catgtgtccg tgtgttgggt
     2521 acgcgtggta ccctgcagga aggaagctga gttggctgct gccaccgctg agcaataact
     2581 agtaattact agcataaccc cttggggcct ctaaacgggt cttgaggggg ttttttgctg
     2641 aaaggaggac agctgatgat tgtcatgctt gccatctgtt ttcttgcaag gtcagaggaa
     2701 ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     2761 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     2821 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     2881 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     2941 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3001 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3061 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3121 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3181 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3241 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3301 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3361 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3421 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3481 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3541 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3601 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3661 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaggatc ctttgatctt
     3721 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3781 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3841 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3901 tatctcagcg atctgtctag ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3961 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4021 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4081 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4141 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4201 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     4261 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     4321 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4381 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4441 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4501 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4561 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     4621 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4681 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4741 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4801 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
//
