LOCUS       sequence-127550-        5275 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for cell-free expression with C-term eGFP
            8xHis tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5275)
  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 5275)
  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_G04
FEATURES             Location/Qualifiers
     source          1..5275
                     /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"
     primer_bind     complement(2073..2094)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2334..2353)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2681..2702
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2754..2777
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      3000..3046
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3124..3140)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3124..3140)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3137..3159)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3148..3164
                     /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(3172..3178,3179..3196,3197..3202))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3217..3238
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3355..3372)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3526..4114)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3606..3625)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4285..5076,5077..5145))
                     /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     4908..4927
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5146..5250)
                     /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 ggcccgaagg
     2041 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     2101 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     2161 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     2221 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     2281 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     2341 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     2401 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     2461 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     2521 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     2581 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     2641 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     2701 tgaccgccgc cgggatcact ctcggcatgg acgagctata caagacctct ggtcaccacc
     2761 atcaccacca tcaccactaa gcggccgccc tcctcctcct ttcttgttcc tttcacgtcg
     2821 ccttctcggt tgtagctggc agacgacgag tcttactttt acgtgtactt ctctatagat
     2881 gatgtatgat ctctctgcat gcgtgttcgt gcatgtgtcc gtgtgttggg tacgcgtggt
     2941 accctgcagg aaggaagctg agttggctgc tgccaccgct gagcaataac tagtaattac
     3001 tagcataacc ccttggggcc tctaaacggg tcttgagggg gttttttgct gaaaggagga
     3061 cagctgatga ttgtcatgct tgccatctgt tttcttgcaa ggtcagagga attcgtaatc
     3121 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     3181 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     3241 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3301 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     3361 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3421 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3481 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3541 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3601 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3661 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3721 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3781 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3841 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3901 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3961 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4021 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4081 gcagcagatt acgcgcagaa aaaaaggatc tcaagaggat cctttgatct tttctacggg
     4141 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     4201 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     4261 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4321 gatctgtcta gttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4381 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4441 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4501 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4561 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4621 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4681 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4741 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4801 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4861 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4921 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4981 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5041 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5101 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     5161 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     5221 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacct
//
