LOCUS       sequence-370469-        6248 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 6248)
  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 6248)
  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 WWP1
FEATURES             Location/Qualifiers
     source          1..6248
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             163..186
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             193..903
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             193..903
                     /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(232..253)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(493..512)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     840..861
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             904..927
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    928..952
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3719..3743)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      3967..4013
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4091..4107)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4091..4107)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4104..4126)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4115..4131
                     /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(4139..4145,4146..4163,4164..4169))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4184..4205
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4322..4339)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4493..5081)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4573..4592)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5252..6043,6044..6112))
                     /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     5875..5894
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6113..6217)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 taatacgact cactataggg acatcttaag tttattttat tttattttat tttattttat
       61 tttattttat tttattttat tttattttat ttaaccatga cagtaatgta taaagtctgt
      121 aaagacatta aacacgtaag tgaaaccatg ggcacctctg gtcaccacca tcaccaccat
      181 caccacggca caagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      241 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
      301 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
      361 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
      421 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
      481 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
      541 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
      601 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
      661 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
      721 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
      781 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
      841 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctatac
      901 aagctggagg tgctcttcca gggtccgaca agtttgtaca aaaaagttgg catggccact
      961 gcttcaccaa ggtctgatac tagtaataac cacagtggaa ggttgcagtt acaggtaact
     1021 gtttctagtg ccaaacttaa aagaaaaaag aactggttcg gaacagcaat atatacagaa
     1081 gtagttgtag atggagaaat tacgaaaaca gcaaaatcca gtagttcttc taatccaaaa
     1141 tgggatgaac agctaactgt aaatgttacg ccacagacta cattggaatt tcaagtttgg
     1201 agccatcgca ctttaaaagc agatgcttta ttaggaaaag caacgataga tttgaaacaa
     1261 gctctgttga tacacaatag aaaattggaa agagtgaaag aacaattaaa actttccttg
     1321 gaaaacaaga atggcatagc acaaactggt gaattgacag ttgtgcttga tggattggtg
     1381 attgagcaag aaaatataac aaactgcagc tcatctccaa ccatagaaat acaggaaaat
     1441 ggtgatgcct tacatgaaaa tggagagcct tcagcaagga caactgccag gttggctgtt
     1501 gaaggcacga atggaataga taatcatgta cctacaagca ctctagtcca aaactcatgc
     1561 tgctcgtatg tagttaatgg agacaacaca ccttcatctc cgtctcaggt tgctgccaga
     1621 cccaaaaata caccagctcc aaaaccactc gcatctgagc ctgccgatga cactgttaat
     1681 ggagaatcat cctcatttgc accaactgat aatgcgtctg tcacgggtac tccagtagtg
     1741 tctgaagaaa atgccttgtc tccaaattgc actagtacta ctgttgaaga tcctccagtt
     1801 caagaaatac tgacttcctc agaaaacaat gaatgtattc cttctaccag tgcagaattg
     1861 gaatctgaag ctagaagtat attagagcct gacacctcta attctagaag tagttctgct
     1921 tttgaagcag ccaaatcaag acagccagat gggtgtatgg atcctgtacg gcagcagtct
     1981 gggaatgcca acacagaaac cttgccatca gggtgggaac aaagaaaaga tcctcatggt
     2041 agaacctatt atgtggatca taatactcga actaccacat gggagagacc acaaccttta
     2101 cctccaggtt gggaaagaag agttgatgat cgtagaagag tttattatgt ggatcataac
     2161 accagaacaa caacgtggca gcggcctacc atggaatctg tccgaaattt tgaacagtgg
     2221 caatctcagc ggaaccaatt gcagggagct atgcaacagt ttaaccaacg atacctctat
     2281 tcggcttcaa tgttagctgc agaaaatgac ccttatggac ctttgccacc aggctgggaa
     2341 aaaagagtgg attcaacaga cagggtttac tttgtgaatc ataacacaaa aacaacccag
     2401 tgggaagatc caagaactca aggcttacag aatgaagaac ccctgccaga aggctgggaa
     2461 attagatata ctcgtgaagg tgtaaggtac tttgttgatc ataacacaag aacaacaaca
     2521 ttcaaagatc ctcgcaatgg gaagtcatct gtaactaaag gtggtccaca aattgcttat
     2581 gaacgcggct ttaggtggaa gcttgctcac ttccgttatt tgtgccagtc taatgcacta
     2641 cctagtcatg taaagatcaa tgtgtcccgg cagacattgt ttgaagattc cttccaacag
     2701 attatggcat taaaacccta tgacttgagg aggcgcttat atgtaatatt tagaggagaa
     2761 gaaggacttg attatggtgg cctagcgaga gaatggtttt tcttgctttc acatgaagtt
     2821 ttgaacccaa tgtattgctt atttgagtat gcgggcaaga acaactattg tctgcagata
     2881 aatccagcat caaccattaa tccagaccat ctttcatact tctgtttcat tggtcgtttt
     2941 attgccatgg cactatttca tggaaagttt atcgatactg gtttctcttt accattctac
     3001 aagcgtatgt taagtaaaaa acttactatt aaggatttgg aatctattga tactgaattt
     3061 tataactccc ttatctggat aagagataac aacattgaag aatgtggctt agaaatgtac
     3121 ttttctgttg acatggagat tttgggaaaa gttacttcac atgacctgaa gttgggaggt
     3181 tccaatattc tggtgactga ggagaacaaa gatgaatata ttggtttaat gacagaatgg
     3241 cgtttttctc gaggagtaca agaacagacc aaagctttcc ttgatggttt taatgaagtt
     3301 gttcctcttc agtggctaca gtacttcgat gaaaaagaat tagaggttat gttgtgtggc
     3361 atgcaggagg ttgacttggc agattggcag agaaatactg tttatcgaca ttatacaaga
     3421 aacagcaagc aaatcatttg gttttggcag tttgtgaaag agacagacaa tgaagtaaga
     3481 atgcgactat tgcagttcgt cactggaacc tgccgtttac ctctaggagg atttgctgag
     3541 ctcatgggaa gtaatgggcc tcaaaagttt tgcattgaaa aagttggcaa agacacttgg
     3601 ttaccaagaa gccatacatg ttttaatcgc ttggatctac caccatataa gagttatgaa
     3661 caactaaagg aaaaacttct ttttgcaata gaagagacag agggatttgg acaagaatgc
     3721 ccaactttct tgtacaaagt ggtgtaagcg gccgccctcc tcctcctttc ttgttccttt
     3781 cacgtcgcct tctcggttgt agctggcaga cgacgagtct tacttttacg tgtacttctc
     3841 tatagatgat gtatgatctc tctgcatgcg tgttcgtgca tgtgtccgtg tgttgggtac
     3901 gcgtggtacc ctgcaggaag gaagctgagt tggctgctgc caccgctgag caataactag
     3961 taattactag cataacccct tggggcctct aaacgggtct tgagggggtt ttttgctgaa
     4021 aggaggacag ctgatgattg tcatgcttgc catctgtttt cttgcaaggt cagaggaatt
     4081 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4141 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4201 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4261 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4321 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4381 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4441 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4501 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4561 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4621 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4681 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4741 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4801 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4861 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4921 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4981 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5041 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaggatcct ttgatctttt
     5101 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5161 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5221 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5281 tctcagcgat ctgtctagtt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5341 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5401 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5461 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5521 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5581 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5641 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5701 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5761 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5821 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5881 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5941 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6001 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6061 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6121 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6181 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6241 ctgacgtc
//
