LOCUS       sequence-272579-        4820 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 4820)
  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 4820)
  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 CYP2D6
FEATURES             Location/Qualifiers
     source          1..4820
                     /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"
     regulatory      2228..2237
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2291..2315)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      2539..2585
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2663..2679)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2663..2679)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2676..2698)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2687..2703
                     /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(2711..2717,2718..2735,2736..2741))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2756..2777
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2894..2911)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3065..3653)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3145..3164)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3824..4615,4616..4684))
                     /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     4447..4466
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4685..4789)
                     /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 caccatgggg
      961 ctagaagcac tggtgcccct ggccatgata gtggccatct tcctgctcct ggtggacctg
     1021 atgcaccggc gccaacgctg ggctgcacgc tacccaccag gccccctgcc actgcccggg
     1081 ctgggcaacc tgctgcatgt ggacttccag aacacaccat actgcttcga ccagttgcgg
     1141 cgccgcttcg gggacgtgtt cagcctgcag ctggcctgga cgccggtggt cgtgctcaat
     1201 gggctggcgg ccgtgcgcga ggcgctggtg acccacggcg aggacaccgc cgaccgcccg
     1261 cctgtgccca tcacccagat cctgggtttc gggccgcgtt cccaaggacg cccctttcgc
     1321 cccaacggtc tcttggacaa agccgtgagc aacgtgatcg cctccctcac ctgcgggcgc
     1381 cgcttcgagt acgacgaccc tcgcttcctc aggctgctgg acctagctca ggagggactg
     1441 aaggaggagt cgggctttct gcgcgaggtg ctgaatgctg tccccgtcct cctgcatatc
     1501 ccagcgctgg ctggcaaggt cctacgcttc caaaaggctt tcctgaccca gctggatgag
     1561 ctgctaactg agcacaggat gacctgggac ccagcccagc ccccccgaga cctgactgag
     1621 gccttcctgg cagagatgga gaaggccaag gggaaccctg agagcagctt caatgatgag
     1681 aacctgtgca tagtggtggc tgacctgttc tctgccggga tggtgaccac ctcgaccacg
     1741 ctggcctggg gcctcctgct catgatccta catccggatg tgcagcgccg tgtccaacag
     1801 gagatcgacg acgtgatagg gcaggtgcgg cgaccagaga tgggtgacca ggctcacatg
     1861 ccctacacca ctgccgtgat tcatgaggtg cagcgctttg gggacatcgt ccccctgggt
     1921 gtgacccata tgacatcccg tgacatcgaa gtacagggct tccgcatccc taagggaacg
     1981 acactcatca ccaacctgtc atcggtgctg aaggatgagg ccgtctggga gaagcccttc
     2041 cgcttccacc ccgaacactt cctggatgcc cagggccact ttgtgaagcc ggaggccttc
     2101 ctgcctttct cagcaggccg ccgtgcatgc ctcggggagc ccctggcccg catggagctc
     2161 ttcctcttct tcacctccct gctgcagcac ttcagcttct cggtgcccac tggacagccc
     2221 cggcccagcc accatggtgt ctttgctttc ctggtgaccc catcccccta tgagctttgt
     2281 gctgtgcccc gcccaacttt cttgtacaaa gtggtgtaag cggccgccct cctcctcctt
     2341 tcttgttcct ttcacgtcgc cttctcggtt gtagctggca gacgacgagt cttactttta
     2401 cgtgtacttc tctatagatg atgtatgatc tctctgcatg cgtgttcgtg catgtgtccg
     2461 tgtgttgggt acgcgtggta ccctgcagga aggaagctga gttggctgct gccaccgctg
     2521 agcaataact agtaattact agcataaccc cttggggcct ctaaacgggt cttgaggggg
     2581 ttttttgctg aaaggaggac agctgatgat tgtcatgctt gccatctgtt ttcttgcaag
     2641 gtcagaggaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     2701 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     2761 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     2821 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     2881 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     2941 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     3001 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     3061 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3121 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3181 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3241 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3301 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3361 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3421 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3481 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     3541 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3601 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaggatc
     3661 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3721 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3781 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3841 gtgaggcacc tatctcagcg atctgtctag ttcgttcatc catagttgcc tgactccccg
     3901 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3961 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     4021 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4081 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     4141 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     4201 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     4261 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     4321 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     4381 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     4441 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4501 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4561 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4621 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4681 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4741 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4801 gaaaagtgcc acctgacgtc
//
