LOCUS       sequence-127545-        5284 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for cell-free expression with N-term 
            8xHis eGFP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5284)
  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 5284)
  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_G03
FEATURES             Location/Qualifiers
     source          1..5284
                     /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..1058
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1105..1410)
                     /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(1207..1226)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1752..2411)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2326..2345
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2412..2514)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2661..2785)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      3009..3055
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3133..3149)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3133..3149)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3146..3168)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3157..3173
                     /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(3181..3187,3188..3205,3206..3211))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3226..3247
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3364..3381)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3535..4123)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3615..3634)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4294..5085,5086..5154))
                     /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     4917..4936
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5155..5259)
                     /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 agctgaacga
      961 gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact
     1021 acataatact gtaaaacaca acatatccag tcactatggg gcccgtcgac ctgcagactg
     1081 gctgtgtata agggagcctg acatttatat tccccagaac atcaggttaa tggcgttttt
     1141 gatgtcattt tcgcggtggc tgagatcagc cacttcttcc ccgataacgg agaccggcac
     1201 actggccata tcggtggtca tcatgcgcca gctttcatcc ccgatatgca ccaccgggta
     1261 aagttcacgg gagactttat ctgacagcag acgtgcactg gccaggggga tcaccatccg
     1321 tcgcccgggc gtgtcaataa tatcactctg tacatccaca aacagacgat aacggctctc
     1381 tcttttatag gtgtaaacct taaactgcat ttcaccagtc cctgttctcg tcagcaaaag
     1441 agccgttcat ttcaataaac cgggcgacct cagccatccc ttcctgattt tccgctttcc
     1501 agcgttcggc acgcagacga cgggcttcat tctgcatggt tgtgcttacc agaccggaga
     1561 tattgacatc atatatgcct tgagcaactg atagctgtcg ctgtcaactg tcactgtaat
     1621 acgctgcttc atagcacacc tctttttgac atacttcggg tatacatatc agtatatatt
     1681 cttataccgc aaaaatcagc gcgcaaatac gcatactgtt atctggcttt tagtaagccg
     1741 gatccacgcg tttacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc
     1801 attctgccga catggaagcc atcacagacg gcatgatgaa cctgaatcgc cagcggcatc
     1861 agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg
     1921 tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg
     1981 aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc
     2041 acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc
     2101 gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat
     2161 atcaccagct caccgtcttt cattgccata cggaattccg gatgagcatt catcaggcgg
     2221 gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa
     2281 aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat
     2341 gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt
     2401 tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa aaatacgccc
     2461 ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccg atcaacgtct
     2521 cattttcgcc aaaagttggc ccagggcttc ccggtatcaa cagggacacc aggatttatt
     2581 tattctgcga agtgatcttc cgtcacaggt atttattcgg cgcaaagtgc gtcgggtgat
     2641 gctgccaact tagcaagctt catagtgact ggatatgttg tgttttacag tattatgtag
     2701 tctgtttttt atgcaaaatc taatttaata tattgatatt tatatcattt tacgtttctc
     2761 gttcagcttt cttgtacaaa gtggtgtaag cggccgccct cctcctcctt tcttgttcct
     2821 ttcacgtcgc cttctcggtt gtagctggca gacgacgagt cttactttta cgtgtacttc
     2881 tctatagatg atgtatgatc tctctgcatg cgtgttcgtg catgtgtccg tgtgttgggt
     2941 acgcgtggta ccctgcagga aggaagctga gttggctgct gccaccgctg agcaataact
     3001 agtaattact agcataaccc cttggggcct ctaaacgggt cttgaggggg ttttttgctg
     3061 aaaggaggac agctgatgat tgtcatgctt gccatctgtt ttcttgcaag gtcagaggaa
     3121 ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     3181 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3241 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3301 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3361 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3421 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3481 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3541 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3601 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3661 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3721 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3781 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3841 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3901 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3961 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4021 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4081 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaggatc ctttgatctt
     4141 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4201 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4261 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4321 tatctcagcg atctgtctag ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4381 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4441 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4501 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4561 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4621 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     4681 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     4741 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4801 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4861 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4921 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4981 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5041 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5101 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5161 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5221 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5281 acct
//
