LOCUS       sequence-147286-        5178 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for cell-free expression with N-term 
            8xHis sfGFP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5178)
  AUTHORS   Gagoski D, Mureev S, Giles N, Johnston W, Dahmer-Heath M, Skalamera 
            D, Gonda TJ, Alexandrov K
  TITLE     Gateway-compatible vectors for high-throughput protein expression in
            pro- and eukaryotic cell-free systems.
  JOURNAL   J Biotechnol. 2015 Feb 10;195:1-7. doi: 
            10.1016/j.jbiotec.2014.12.006. Epub 2014 Dec 19.
  PUBMED    25529348
REFERENCE   2  (bases 1 to 5178)
  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: "J 
            Biotechnol."; date: "2015-02-10"; volume: "195:1-7. doi"; pages: " 
            10.1016/j.jbiotec.2014.12.006. Epub 2014 Dec 19"
COMMENT     Sequence Label: pCellFree_G09
FEATURES             Location/Qualifiers
     source          1..5178
                     /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             join(199..201,202..912)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             199..912
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(229..257)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     852..872
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             913..936
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    937..1061
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1108..1413)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(1210..1229)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1755..2414)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2329..2348
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2415..2517)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      2903..2949
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3027..3043)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3027..3043)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3040..3062)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3051..3067
                     /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(3075..3081,3082..3099,3100..3105))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3120..3141
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3258..3275)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3429..4017)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3509..3528)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4188..4979,4980..5048))
                     /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     4811..4830
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5049..5153)
                     /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 acggcacaat gagcaaagga gaagaacttt tcactggagt tgtcccaatt
      241 cttgttgaat tagatggtga tgttaatggg cacaaatttt ctgtccgtgg agagggtgaa
      301 ggtgatgcta caaacggaaa actcaccctt aaatttattt gcactactgg aaaactacct
      361 gttccgtggc caacacttgt cactactctg acctatggtg ttcaatgctt ttcccgttat
      421 ccggatcaca tgaaacggca tgactttttc aagagtgcca tgcccgaagg ttatgtacag
      481 gaacgcacta tatctttcaa agatgacggg acctacaaga cgcgtgctga agtcaagttt
      541 gaaggtgata cccttgttaa tcgtatcgag ttaaagggta ttgattttaa agaagatgga
      601 aacattcttg gacacaaact cgagtacaac tttaactcac acaatgtata catcacggca
      661 gacaaacaaa agaatggaat caaagctaac ttcaaaattc gccacaacgt tgaagatggt
      721 tccgttcaac tagcagacca ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt
      781 ttaccagaca accattacct gtcgacacaa tctgtccttt cgaaagatcc caacgaaaag
      841 cgtgaccaca tggtccttct tgagtttgta actgctgctg ggattacaca tggcatggat
      901 gagctctaca aactggaggt gctcttccag ggtccgacaa gtttgtacaa aaaagctgaa
      961 cgagaaacgt aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag
     1021 actacataat actgtaaaac acaacatatc cagtcactat ggggcccgtc gacctgcaga
     1081 ctggctgtgt ataagggagc ctgacattta tattccccag aacatcaggt taatggcgtt
     1141 tttgatgtca ttttcgcggt ggctgagatc agccacttct tccccgataa cggagaccgg
     1201 cacactggcc atatcggtgg tcatcatgcg ccagctttca tccccgatat gcaccaccgg
     1261 gtaaagttca cgggagactt tatctgacag cagacgtgca ctggccaggg ggatcaccat
     1321 ccgtcgcccg ggcgtgtcaa taatatcact ctgtacatcc acaaacagac gataacggct
     1381 ctctctttta taggtgtaaa ccttaaactg catttcacca gtccctgttc tcgtcagcaa
     1441 aagagccgtt catttcaata aaccgggcga cctcagccat cccttcctga ttttccgctt
     1501 tccagcgttc ggcacgcaga cgacgggctt cattctgcat ggttgtgctt accagaccgg
     1561 agatattgac atcatatatg ccttgagcaa ctgatagctg tcgctgtcaa ctgtcactgt
     1621 aatacgctgc ttcatagcac acctcttttt gacatacttc gggtatacat atcagtatat
     1681 attcttatac cgcaaaaatc agcgcgcaaa tacgcatact gttatctggc ttttagtaag
     1741 ccggatccac gcgtttacgc cccgccctgc cactcatcgc agtactgttg taattcatta
     1801 agcattctgc cgacatggaa gccatcacag acggcatgat gaacctgaat cgccagcggc
     1861 atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag
     1921 ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag
     1981 acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac
     2041 gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag
     2101 agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc
     2161 catatcacca gctcaccgtc tttcattgcc atacggaatt ccggatgagc attcatcagg
     2221 cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt
     2281 aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga
     2341 aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg
     2401 atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc aaaaaatacg
     2461 cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg ccgatcaacg
     2521 tctcattttc gccaaaagtt ggcccagggc ttcccggtat caacagggac accaggattt
     2581 atttattctg cgaagtgatc ttccgtcaca ggtatttatt cggcgcaaag tgcgtcgggt
     2641 gatgctgcca acttagcaag ctttcttgta caaagtggtg taagcggccg ccctcctcct
     2701 cctttcttgt tcctttcacg tcgccttctc ggttgtagct ggcagacgac gagtcttact
     2761 tttacgtgta cttctctata gatgatgtat gatctctctg catgcgtgtt cgtgcatgtg
     2821 tccgtgtgtt gggtacgcgt ggtaccctgc aggaaggaag ctgagttggc tgctgccacc
     2881 gctgagcaat aactagtaat tactagcata accccttggg gcctctaaac gggtcttgag
     2941 ggggtttttt gctgaaagga ggacagctga tgattgtcat gcttgccatc tgttttcttg
     3001 caaggtcaga ggaattcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3061 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3121 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3181 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3241 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3301 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3361 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3421 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3481 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3541 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3601 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3661 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3721 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3781 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3841 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3901 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3961 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagag
     4021 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4081 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4141 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4201 atcagtgagg cacctatctc agcgatctgt ctagttcgtt catccatagt tgcctgactc
     4261 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4321 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4381 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4441 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4501 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4561 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4621 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4681 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4741 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4801 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4861 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4921 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4981 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5041 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5101 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5161 ccccgaaaag tgccacct
//
