LOCUS       sequence-365619-        4838 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 4838)
  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 4838)
  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 TFAP2B
FEATURES             Location/Qualifiers
     source          1..4838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     18..37
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        18..36
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             180..203
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             210..920
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             210..920
                     /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(249..270)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(510..529)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     857..878
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             921..944
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    945..969
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2326..2350)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      2574..2620
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2698..2714)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2698..2714)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2711..2733)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2722..2738
                     /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(2746..2752,2753..2770,2771..2776))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2791..2812
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2929..2946)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3100..3688)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3180..3199)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3859..4650,4651..4719))
                     /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     4482..4501
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4720..4824)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aagtgccacc tgacgtctaa tacgactcac tatagggaca tcttaagttt attttatttt
       61 attttatttt attttatttt attttatttt attttatttt attttattta accatgacag
      121 taatgtataa agtctgtaaa gacattaaac acgtaagtga aaccatgggc acctctggtc
      181 accaccatca ccaccatcac cacggcacaa gcaagggcga ggagctgttc accggggtgg
      241 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
      301 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
      361 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
      421 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
      481 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
      541 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
      601 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
      661 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
      721 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
      781 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
      841 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
      901 gcatggacga gctatacaag ctggaggtgc tcttccaggg tccgacaagt ttgtacaaaa
      961 aagcaggctc caccatgctc tggaagcttg tggagaatgt caagtacgaa gatatctatg
     1021 aggaccggca cgatggtgtc ccgagccaca gctcgcggct ctcccagctg ggctcggtgt
     1081 cccaaggacc ctactcgagc gccccgccgc tgtcccacac cccgtcgtcg gacttccagc
     1141 cgccctactt cccacccccc taccagccgc tcccctacca ccagagccag gacccctact
     1201 cccacgtcaa cgacccctac tccctgaacc cactgcacca gccccagcaa catccctggg
     1261 ggcaacggca gcggcaagaa gtgggttcgg aagccggctc tctcctgccc cagcctcggg
     1321 ccgccttgcc ccagctctcg ggccttgacc cccggaggga ctaccactcg gtccgccggc
     1381 cggacgtgct gctgcattcg gcgcaccacg gcctggacgc gggcatgggt gacagcctct
     1441 cgctgcacgg cctcggccat cccggaatgg aagacgtcca gtcagttgaa gatgccaata
     1501 acagcggcat gaatctattg gaccagtctg tcattaaaaa agttccagtt cctcccaaat
     1561 cggtgacttc tctaatgatg aataaagacg gcttcctggg aggcatgtct gtcaacaccg
     1621 gcgaggtgtt ttgctccgtc ccaggccgtt tgtctctgct cagttcaact tcgaagtaca
     1681 aagtaactgt gggagaagtt cagagacggc tgtcgccccc tgaatgcctc aatgcatctc
     1741 tcctcggcgg agtcctcaga agagccaaat cgaaaaatgg ggggagatct ttgcgagaaa
     1801 ggctagaaaa aatcggtttg aatttacccg cgggcaggcg caaagcagca aatgtcacgt
     1861 tactcacctc cctggtggaa ggagaagctg ttcacttagc tagggatttt gggtacattt
     1921 gcgaaacgga gtttcccgcc aaagccgtct ctgagtattt gaaccggcag cacacagacc
     1981 cgagtgacct gcactcccga aagaatatgc tgttggccac caagcaactt tgtaaagaat
     2041 ttacggatct actggcgcag gaccggacac cgatagggaa cagccgaccc agccccatcc
     2101 tggagccggg gatccagagc tgcctcacgc acttcagcct catcacgcac ggcttcggcg
     2161 ccccggccat ttgcgccgcg ctcacggccc tgcagaacta tctcaccgag gcgctcaaag
     2221 gcatggacaa gatgttcttg aacaacacca ccactaacag gcacacgtct ggggaaggcc
     2281 caggtagtaa aactggcgac aaggaggaga aacacaggaa attggaccca gctttcttgt
     2341 acaaagtggt gtaagcggcc gccctcctcc tcctttcttg ttcctttcac gtcgccttct
     2401 cggttgtagc tggcagacga cgagtcttac ttttacgtgt acttctctat agatgatgta
     2461 tgatctctct gcatgcgtgt tcgtgcatgt gtccgtgtgt tgggtacgcg tggtaccctg
     2521 caggaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagtaa ttactagcat
     2581 aaccccttgg ggcctctaaa cgggtcttga gggggttttt tgctgaaagg aggacagctg
     2641 atgattgtca tgcttgccat ctgttttctt gcaaggtcag aggaattcgt aatcatggtc
     2701 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     2761 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     2821 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     2881 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     2941 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3001 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3061 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3121 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3181 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3241 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3301 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3361 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3421 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3481 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3541 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3601 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3661 gattacgcgc agaaaaaaag gatctcaaga ggatcctttg atcttttcta cggggtctga
     3721 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     3781 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3841 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3901 tctagttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3961 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4021 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4081 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4141 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4201 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4261 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4321 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4381 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4441 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4501 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4561 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4621 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4681 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     4741 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4801 aaataaacaa ataggggttc cgcgcacatt tccccgaa
//
