LOCUS       sequence-272580-        4991 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 4991)
  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 4991)
  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 CYP3A4
FEATURES             Location/Qualifiers
     source          1..4991
                     /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    complement(2462..2486)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      2710..2756
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2834..2850)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2834..2850)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2847..2869)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2858..2874
                     /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(2882..2888,2889..2906,2907..2912))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2927..2948
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3065..3082)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3236..3824)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3316..3335)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3995..4786,4787..4855))
                     /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     4618..4637
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4856..4960)
                     /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 agtttgcaca aaaaagttgg catggctctc
      961 atcccagact tggccatgga aacctggctt ctcctggctg tcagcctggt gctcctctat
     1021 ctatatggaa cccattcaca tggacttttt aagaagcttg gaattccagg gcccacacct
     1081 ctgccttttt tgggaaatat tttgtcctac cataagggct tttgtatgtt tgacatggaa
     1141 tgtcataaaa agtatggaaa agtgtggggc ttttatgatg gtcaacagcc tgtgctggct
     1201 atcacagatc ctgacatgat caaaacagtg ctagtgaaag aatgttattc tgtcttcaca
     1261 aaccggaggc cttttggtcc agtgggattt atgaaaagtg ccatctctat agctgaggat
     1321 gaagaatgga agagattacg atcattgctg tctccaacct tcaccagtgg aaaactcaag
     1381 gagatggtcc ctatcattgc ccagtatgga gatgtgttgg tgagaaatct gaggcgggaa
     1441 gcagagacag gcaagcctgt caccttgaaa gacgtctttg gggcctacag catggatgtg
     1501 atcactagca catcatttgg agtgaacatc gactctctca acaatccaca agaccccttt
     1561 gtggaaaaca ccaagaagct tttaagattt gattttttgg atccattctt tctctcaata
     1621 acagtctttc cattcctcat cccaattctt gaagtattaa atatctgtgt gtttccaaga
     1681 gaagttacaa attttttaag aaaatctgta aaaaggatga aagaaagtcg cctcgaagat
     1741 acacaaaagc accgagtgga tttccttcag ctgatgattg actctcagaa ttcaaaagaa
     1801 actgagtccc acaaagctct gtccgatctg gagctcgtgg cccaatcaat tatctttatt
     1861 tttgctggct atgaaaccac gagcagtgtt ctctccttca ttatgtatga actggccact
     1921 caccctgatg tccagcagaa actgcaggag gaaattgatg cagttttacc caataaggca
     1981 ccacccacct atgatactgt gctacagatg gagtatcttg acatggtggt gaatgaaacg
     2041 ctcagattat tcccaattgc tatgagactt gagagggtct gcaaaaaaga tgttgagatc
     2101 aatgggatgt tcattcccaa aggggtggtg gtgatgattc caagctatgc tcttcaccgt
     2161 gacccaaagt actggacaga gcctgagaag ttcctccctg aaagattcag caagaagaac
     2221 aaggacaaca tagatcctta catatacaca ccctttggaa gtggacccag aaactgcatt
     2281 ggcatgaggt ttgctctcat gaacatgaaa cttgctctaa tcagagtcct tcagaacttc
     2341 tccttcaaac cttgtaaaga aacacagatc cccctgaaat taagcttagg aggacttctt
     2401 caaccagaaa aacccgttgt tctaaaggtt gagtcaaggg atggcaccgt aagtggagcc
     2461 tgcccaactt tcttgtacaa agtggtgtaa gcggccgccc tcctcctcct ttcttgttcc
     2521 tttcacgtcg ccttctcggt tgtagctggc agacgacgag tcttactttt acgtgtactt
     2581 ctctatagat gatgtatgat ctctctgcat gcgtgttcgt gcatgtgtcc gtgtgttggg
     2641 tacgcgtggt accctgcagg aaggaagctg agttggctgc tgccaccgct gagcaataac
     2701 tagtaattac tagcataacc ccttggggcc tctaaacggg tcttgagggg gttttttgct
     2761 gaaaggagga cagctgatga ttgtcatgct tgccatctgt tttcttgcaa ggtcagagga
     2821 attcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2881 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     2941 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3001 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3061 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3121 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3181 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3241 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3301 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3361 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3421 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3481 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3541 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3601 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3661 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3721 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3781 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaggat cctttgatct
     3841 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3901 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3961 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4021 ctatctcagc gatctgtcta gttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4081 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4141 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4201 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4261 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4321 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4381 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4441 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4501 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4561 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4621 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4681 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4741 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4801 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4861 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4921 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4981 cacctgacgt c
//
