LOCUS       sequence-127548-        4534 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for cell-free expression with C-term 
            8xHis tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4534)
  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 4534)
  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_G02
FEATURES             Location/Qualifiers
     source          1..4534
                     /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="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      2259..2305
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2383..2399)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2383..2399)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2396..2418)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2407..2423
                     /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(2431..2437,2438..2455,2456..2461))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2476..2497
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2614..2631)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2785..3373)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2865..2884)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3544..4335,4336..4404))
                     /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     4167..4186
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4405..4509)
                     /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 tgcaccacca tcaccaccat caccactaag
     2041 cggccgccct cctcctcctt tcttgttcct ttcacgtcgc cttctcggtt gtagctggca
     2101 gacgacgagt cttactttta cgtgtacttc tctatagatg atgtatgatc tctctgcatg
     2161 cgtgttcgtg catgtgtccg tgtgttgggt acgcgtggta ccctgcagga aggaagctga
     2221 gttggctgct gccaccgctg agcaataact agtaattact agcataaccc cttggggcct
     2281 ctaaacgggt cttgaggggg ttttttgctg aaaggaggac agctgatgat tgtcatgctt
     2341 gccatctgtt ttcttgcaag gtcagaggaa ttcgtaatca tggtcatagc tgtttcctgt
     2401 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     2461 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     2521 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     2581 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     2641 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     2701 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     2761 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     2821 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     2881 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     2941 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3001 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3061 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3121 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3181 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     3241 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3301 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3361 aaaaggatct caagaggatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3421 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     3481 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3541 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctag ttcgttcatc
     3601 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3661 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     3721 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3781 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     3841 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3901 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     3961 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4021 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4081 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4141 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     4201 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     4261 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     4321 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     4381 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     4441 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     4501 ggttccgcgc acatttcccc gaaaagtgcc acct
//
