LOCUS       sequence-243807-        4796 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 4796)
  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 4796)
  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 WAVE 2
FEATURES             Location/Qualifiers
     source          1..4796
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     7..26
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7..25
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             169..192
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             199..909
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             199..909
                     /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(238..259)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(499..518)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     846..867
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             910..933
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    934..958
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2273..2297)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      2521..2567
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2645..2661)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2645..2661)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2658..2680)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2669..2685
                     /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(2693..2699,2700..2717,2718..2723))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2738..2759
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2876..2893)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3047..3635)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3127..3146)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3806..4597,4598..4666))
                     /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     4429..4448
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4667..4771)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtctaat acgactcact atagggacat cttaagttta ttttatttta ttttatttta
       61 ttttatttta ttttatttta ttttatttta ttttatttaa ccatgacagt aatgtataaa
      121 gtctgtaaag acattaaaca cgtaagtgaa accatgggca cctctggtca ccaccatcac
      181 caccatcacc acggcacaag caagggcgag gagctgttca ccggggtggt gcccatcctg
      241 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      301 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      361 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      421 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      481 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      541 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
      601 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
      661 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
      721 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
      781 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
      841 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
      901 ctatacaagc tggaggtgct cttccagggt ccgacaagtt tgtacaaaaa agcaggcttc
      961 atgccgttag taacgaggaa catcgagcca aggcacctgt gccgtcagac gttgcctagc
     1021 gttagaagcg agctggaatg cgtgaccaac atcaccctgg caaatgtcat ccgacagctg
     1081 ggcagtctga gtaaatatgc agaggacatt tttggagagc tctttactca ggcaaatacc
     1141 tttgcctctc gggtaagctc ccttgctgag agggtcgacc gactacaggt taaagtcact
     1201 cagctggatc ccaaggaaga agaagtgtca ctgcaaggaa tcaacacccg aaaagccttc
     1261 agaagttcca ccattcaaga ccagaagctt tttgacagaa actctctccc agtgcctgtc
     1321 ttagaaacat acaatacctg tgatactcct ccccctctca acaatcttac cccttacagg
     1381 gacgatggaa aagaggcact caaattctac acagaccctt catacttctt tgatctttgg
     1441 aaggagaaga tgctgcagga caccaaggat atcatgaaag agaagagaaa gcataggaaa
     1501 gaaaagaaag ataatccaaa tcgagggaat gtaaacccac gtaaaatcaa gacacgtaag
     1561 gaagagtggg agaaaatgaa gatggggcaa gaatttgtgg agtccaaaga aaagctgggg
     1621 acttctgggt atccacccac tttggtgtac cagaatggca gcattggctg tgttgaaaac
     1681 gtggatgcaa gtagctatcc gccaccacca cagtcagact ctgcttcttc accttctcct
     1741 tccttctccg aggacaactt gcctcctcca ccagcagaat tcagttaccc agtggacaac
     1801 caaagaggat ctggtttggc tggacccaaa agatccagtg tggtcagccc aagccatcca
     1861 ccaccagctc ctcctctagg ctctccacca ggccctaaac ccgggtttgc tccaccacct
     1921 cccttgtccc agccaacagg aggagcacct cctcctcccc ctcctcctcc tcctccgggg
     1981 ccccctcctc cccctttcac tggtgcagat ggccagcctg ctataccacc accgctttct
     2041 gataccacca agcccaagtc ctccttgcct gccgtgagcg atgcccgtag cgacctgctt
     2101 tcagccatcc gtcaaggttt tcagctgcgc agggttgagg agcagcggga acaagagaag
     2161 cgggatgttg tgggcaatga cgtggccacc atcttgtctc gtcgcattgc tgttgagtac
     2221 agtgactcag aagatgactc ctctgaattt gatgaggacg actggtccga taacccagct
     2281 ttcttgtaca aagtggtgta agcggccgcc ctcctcctcc tttcttgttc ctttcacgtc
     2341 gccttctcgg ttgtagctgg cagacgacga gtcttacttt tacgtgtact tctctataga
     2401 tgatgtatga tctctctgca tgcgtgttcg tgcatgtgtc cgtgtgttgg gtacgcgtgg
     2461 taccctgcag gaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagtaatta
     2521 ctagcataac cccttggggc ctctaaacgg gtcttgaggg ggttttttgc tgaaaggagg
     2581 acagctgatg attgtcatgc ttgccatctg ttttcttgca aggtcagagg aattcgtaat
     2641 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     2701 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     2761 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     2821 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     2881 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     2941 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3001 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3061 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3121 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3181 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3241 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3301 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3361 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3421 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3481 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3541 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3601 agcagcagat tacgcgcaga aaaaaaggat ctcaagagga tcctttgatc ttttctacgg
     3661 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3721 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3781 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3841 cgatctgtct agttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3901 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3961 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4021 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4081 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     4141 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     4201 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4261 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4321 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4381 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     4441 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4501 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4561 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4621 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4681 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4741 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacct
//
