LOCUS       sequence-127547-        4537 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for cell-free expression with N-term 
            8xHis tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4537)
  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 4537)
  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_G01
FEATURES             Location/Qualifiers
     source          1..4537
                     /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             160..183
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     protein_bind    187..311
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(358..663)
                     /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(460..479)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1005..1664)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1579..1598
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1665..1767)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(1914..2038)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      2262..2308
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2386..2402)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2386..2402)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2399..2421)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2410..2426
                     /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(2434..2440,2441..2458,2459..2464))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2479..2500
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2617..2634)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2788..3376)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2868..2887)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3547..4338,4339..4407))
                     /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     4170..4189
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4408..4512)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtctaat acgactcact atagggacat cttaagttta ttttatttta ttttatttta
       61 ttttatttta ttttatttta ttttatttta ttttatttaa ccatgacagt aatgtataaa
      121 gtctgtaaag acattaaaca cgtaagtgaa accatgggcc accaccatca ccaccatcac
      181 cacggcacaa gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat
      241 atattaaatt agattttgca taaaaaacag actacataat actgtaaaac acaacatatc
      301 cagtcactat ggggcccgtc gacctgcaga ctggctgtgt ataagggagc ctgacattta
      361 tattccccag aacatcaggt taatggcgtt tttgatgtca ttttcgcggt ggctgagatc
      421 agccacttct tccccgataa cggagaccgg cacactggcc atatcggtgg tcatcatgcg
      481 ccagctttca tccccgatat gcaccaccgg gtaaagttca cgggagactt tatctgacag
      541 cagacgtgca ctggccaggg ggatcaccat ccgtcgcccg ggcgtgtcaa taatatcact
      601 ctgtacatcc acaaacagac gataacggct ctctctttta taggtgtaaa ccttaaactg
      661 catttcacca gtccctgttc tcgtcagcaa aagagccgtt catttcaata aaccgggcga
      721 cctcagccat cccttcctga ttttccgctt tccagcgttc ggcacgcaga cgacgggctt
      781 cattctgcat ggttgtgctt accagaccgg agatattgac atcatatatg ccttgagcaa
      841 ctgatagctg tcgctgtcaa ctgtcactgt aatacgctgc ttcatagcac acctcttttt
      901 gacatacttc gggtatacat atcagtatat attcttatac cgcaaaaatc agcgcgcaaa
      961 tacgcatact gttatctggc ttttagtaag ccggatccac gcgtttacgc cccgccctgc
     1021 cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa gccatcacag
     1081 acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat
     1141 ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa
     1201 ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta
     1261 gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac
     1321 tgccggaaat cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg
     1381 aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc
     1441 atacggaatt ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa
     1501 aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc
     1561 tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat
     1621 tgggatatat caacggtggt atatccagtg atttttttct ccattttagc ttccttagct
     1681 cctgaaaatc tcgataactc aaaaaatacg cccggtagtg atcttatttc attatggtga
     1741 aagttggaac ctcttacgtg ccgatcaacg tctcattttc gccaaaagtt ggcccagggc
     1801 ttcccggtat caacagggac accaggattt atttattctg cgaagtgatc ttccgtcaca
     1861 ggtatttatt cggcgcaaag tgcgtcgggt gatgctgcca acttagcaag cttcatagtg
     1921 actggatatg ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta
     1981 atatattgat atttatatca ttttacgttt ctcgttcagc tttcttgtac aaagtggtgt
     2041 aagcggccgc cctcctcctc ctttcttgtt cctttcacgt cgccttctcg gttgtagctg
     2101 gcagacgacg agtcttactt ttacgtgtac ttctctatag atgatgtatg atctctctgc
     2161 atgcgtgttc gtgcatgtgt ccgtgtgttg ggtacgcgtg gtaccctgca ggaaggaagc
     2221 tgagttggct gctgccaccg ctgagcaata actagtaatt actagcataa ccccttgggg
     2281 cctctaaacg ggtcttgagg gggttttttg ctgaaaggag gacagctgat gattgtcatg
     2341 cttgccatct gttttcttgc aaggtcagag gaattcgtaa tcatggtcat agctgtttcc
     2401 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     2461 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     2521 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2581 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     2641 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     2701 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     2761 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     2821 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     2881 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     2941 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3001 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3061 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3121 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3181 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     3241 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3301 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     3361 aaaaaaagga tctcaagagg atcctttgat cttttctacg gggtctgacg ctcagtggaa
     3421 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     3481 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     3541 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tagttcgttc
     3601 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     3661 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     3721 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     3781 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     3841 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     3901 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     3961 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4021 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4081 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4141 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4201 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4261 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4321 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     4381 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     4441 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     4501 aggggttccg cgcacatttc cccgaaaagt gccacct
//
