LOCUS       sequence-127551-        5266 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for cell-free expression with C-term 
            mCherry 8xHis tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5266)
  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 5266)
  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_G06
FEATURES             Location/Qualifiers
     source          1..5266
                     /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(2173..2191)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2441..2460
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2745..2768
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      2991..3037
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3115..3131)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3115..3131)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3128..3150)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3139..3155
                     /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(3163..3169,3170..3187,3188..3193))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3208..3229
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3346..3363)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3517..4105)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3597..3616)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4276..5067,5068..5136))
                     /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     4899..4918
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5137..5241)
                     /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 gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg
     2101 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     2161 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca
     2221 tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc
     2281 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     2341 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     2401 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
     2461 ctatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     2521 agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca
     2581 cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg
     2641 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc
     2701 gccactccac cggcggcatg gacgagctgt acaagacctc tggtcaccac catcaccacc
     2761 atcaccacta agcggccgcc ctcctcctcc tttcttgttc ctttcacgtc gccttctcgg
     2821 ttgtagctgg cagacgacga gtcttacttt tacgtgtact tctctataga tgatgtatga
     2881 tctctctgca tgcgtgttcg tgcatgtgtc cgtgtgttgg gtacgcgtgg taccctgcag
     2941 gaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagtaatta ctagcataac
     3001 cccttggggc ctctaaacgg gtcttgaggg ggttttttgc tgaaaggagg acagctgatg
     3061 attgtcatgc ttgccatctg ttttcttgca aggtcagagg aattcgtaat catggtcata
     3121 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     3181 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     3241 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3301 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     3361 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3421 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3481 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3541 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3601 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3661 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3721 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3781 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3841 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3901 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3961 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4021 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4081 tacgcgcaga aaaaaaggat ctcaagagga tcctttgatc ttttctacgg ggtctgacgc
     4141 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4201 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4261 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4321 agttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4381 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     4441 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4501 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4561 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4621 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4681 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4741 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4801 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4861 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4921 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4981 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5041 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5101 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5161 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5221 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacct
//
