LOCUS       sequence-127554-        5272 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for cell-free expression with C-term 
            sfGFP 8xHis tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5272)
  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 5272)
  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_G10
FEATURES             Location/Qualifiers
     source          1..5272
                     /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"
     protein_bind    160..284
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(331..636)
                     /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(433..452)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(978..1637)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1552..1571
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1638..1740)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(1887..2011)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2013..2036
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(2037..2039,2040..2750)
                     /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             2037..2750
                     /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(2067..2095)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2690..2710
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2751..2774
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      2997..3043
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3121..3137)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3121..3137)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3134..3156)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3145..3161
                     /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(3169..3175,3176..3193,3194..3199))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3214..3235
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3352..3369)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3523..4111)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3603..3622)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4282..5073,5074..5142))
                     /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     4905..4924
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5143..5247)
                     /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 caagtttgta caaaaaagct
      181 gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa
      241 cagactacat aatactgtaa aacacaacat atccagtcac tatggggccc gtcgacctgc
      301 agactggctg tgtataaggg agcctgacat ttatattccc cagaacatca ggttaatggc
      361 gtttttgatg tcattttcgc ggtggctgag atcagccact tcttccccga taacggagac
      421 cggcacactg gccatatcgg tggtcatcat gcgccagctt tcatccccga tatgcaccac
      481 cgggtaaagt tcacgggaga ctttatctga cagcagacgt gcactggcca gggggatcac
      541 catccgtcgc ccgggcgtgt caataatatc actctgtaca tccacaaaca gacgataacg
      601 gctctctctt ttataggtgt aaaccttaaa ctgcatttca ccagtccctg ttctcgtcag
      661 caaaagagcc gttcatttca ataaaccggg cgacctcagc catcccttcc tgattttccg
      721 ctttccagcg ttcggcacgc agacgacggg cttcattctg catggttgtg cttaccagac
      781 cggagatatt gacatcatat atgccttgag caactgatag ctgtcgctgt caactgtcac
      841 tgtaatacgc tgcttcatag cacacctctt tttgacatac ttcgggtata catatcagta
      901 tatattctta taccgcaaaa atcagcgcgc aaatacgcat actgttatct ggcttttagt
      961 aagccggatc cacgcgttta cgccccgccc tgccactcat cgcagtactg ttgtaattca
     1021 ttaagcattc tgccgacatg gaagccatca cagacggcat gatgaacctg aatcgccagc
     1081 ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag
     1141 aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct
     1201 gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa
     1261 cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc
     1321 cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta
     1381 tcccatatca ccagctcacc gtctttcatt gccatacgga attccggatg agcattcatc
     1441 aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc
     1501 tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac
     1561 tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca
     1621 gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat
     1681 acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca
     1741 acgtctcatt ttcgccaaaa gttggcccag ggcttcccgg tatcaacagg gacaccagga
     1801 tttatttatt ctgcgaagtg atcttccgtc acaggtattt attcggcgca aagtgcgtcg
     1861 ggtgatgctg ccaacttagc aagcttcata gtgactggat atgttgtgtt ttacagtatt
     1921 atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg
     1981 tttctcgttc agctttcttg tacaaagtgg tgctggaggt gctcttccag ggcccgatga
     2041 gcaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg
     2101 ttaatgggca caaattttct gtccgtggag agggtgaagg tgatgctaca aacggaaaac
     2161 tcacccttaa atttatttgc actactggaa aactacctgt tccgtggcca acacttgtca
     2221 ctactctgac ctatggtgtt caatgctttt cccgttatcc ggatcacatg aaacggcatg
     2281 actttttcaa gagtgccatg cccgaaggtt atgtacagga acgcactata tctttcaaag
     2341 atgacgggac ctacaagacg cgtgctgaag tcaagtttga aggtgatacc cttgttaatc
     2401 gtatcgagtt aaagggtatt gattttaaag aagatggaaa cattcttgga cacaaactcg
     2461 agtacaactt taactcacac aatgtataca tcacggcaga caaacaaaag aatggaatca
     2521 aagctaactt caaaattcgc cacaacgttg aagatggttc cgttcaacta gcagaccatt
     2581 atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt accagacaac cattacctgt
     2641 cgacacaatc tgtcctttcg aaagatccca acgaaaagcg tgaccacatg gtccttcttg
     2701 agtttgtaac tgctgctggg attacacatg gcatggatga gctctacaaa caccaccatc
     2761 accaccatca ccactaagcg gccgccctcc tcctcctttc ttgttccttt cacgtcgcct
     2821 tctcggttgt agctggcaga cgacgagtct tacttttacg tgtacttctc tatagatgat
     2881 gtatgatctc tctgcatgcg tgttcgtgca tgtgtccgtg tgttgggtac gcgtggtacc
     2941 ctgcaggaag gaagctgagt tggctgctgc caccgctgag caataactag taattactag
     3001 cataacccct tggggcctct aaacgggtct tgagggggtt ttttgctgaa aggaggacag
     3061 ctgatgattg tcatgcttgc catctgtttt cttgcaaggt cagaggaatt cgtaatcatg
     3121 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     3181 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     3241 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     3301 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     3361 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3421 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     3481 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3541 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3601 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3661 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3721 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3781 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3841 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3901 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3961 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4021 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4081 gcagattacg cgcagaaaaa aaggatctca agaggatcct ttgatctttt ctacggggtc
     4141 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4201 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4261 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4321 ctgtctagtt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4381 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     4441 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4501 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4561 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     4621 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     4681 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4741 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4801 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4861 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4921 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4981 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5041 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5101 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5161 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5221 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ct
//
