LOCUS       sequence-305941-        6176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free expression vector for Zika virus NS5 protein with C-term 
            mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6176)
  AUTHORS   Varasteh Moradi S, Gagoski D, Mureev S, Walden P, McMahon KA, Parton
            RG, Johnston WA, Alexandrov K
  TITLE     Mapping Interactions among Cell-Free Expressed Zika Virus Proteins.
  JOURNAL   J Proteome Res. 2020 Apr 3;19(4):1522-1532. doi: 
            10.1021/acs.jproteome.9b00771. Epub 2020 Mar 9.
  PUBMED    32081002
REFERENCE   2  (bases 1 to 6176)
  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"; doi: 
            "10.1021/acs.jproteome.9b00771"; journalName: "J Proteome Res"; 
            date: "2020-04-3- 3"; volume: "19"; issue: "4"; pages: "1522-1532"
COMMENT     Sequence Label: pCellFree_G06_ZIKV NS5
FEATURES             Location/Qualifiers
     source          1..6176
                     /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..184
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2897..2921)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2923..2946
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(3083..3101)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3351..3370
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3655..3678
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      3901..3947
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4025..4041)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4025..4041)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4038..4060)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4049..4065
                     /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(4073..4079,4080..4097,4098..4103))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4118..4139
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4256..4273)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4427..5015)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4507..4526)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5186..5977,5978..6046))
                     /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     5809..5828
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6047..6151)
                     /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 caaaaaagca
      181 ggcttcggag gtgggacggg agagactctg ggagagaagt ggaaagctcg tctgaatcag
      241 atgtcggccc tggagttcta ctcttataaa aagtcaggta tcactgaagt gtgtagagag
      301 gaggctcgcc gtgccctcaa ggatggagtg gccacaggag gacatgccgt atcccgggga
      361 agtgcaaaga tcagatggtt ggaggagaga ggatatctgc agccctatgg gaaggttgtt
      421 gacctcggat gtggcagagg gggctggagc tattatgccg ccaccatccg caaagtgcag
      481 gaggtgagag gatacacaaa gggaggtccc ggtcatgaag aacccatgct ggtgcaaagc
      541 tatgggtgga acatagttcg tctcaagagt ggagtggacg tcttccacat ggcggctgag
      601 ccgtgtgaca ctctgctgtg tgacataggt gagtcatcat ctagtcctga agtggaagag
      661 acacgaacac tcagagtgct ctctatggtg ggggactggc ttgaaaaaag accaggggcc
      721 ttctgtataa aggtgctgtg cccatacacc agcactatga tggaaacgat ggagcgactg
      781 caacgtaggc atgggggagg attagtcaga gtgccattgt gtcgcaactc cacacatgag
      841 atgtactggg tctctggggc aaagagcaac atcataaaaa gtgtgtccac cacaagtcag
      901 ctcctcctgg gacgcatgga tggccccagg aggccagtga aatatgagga ggatgtgaac
      961 ctcggctcgg gtacacgagc tgtggcaagc tgtgctgagg ctcctaacat gaaaatcatc
     1021 ggcaggcgca ttgagagaat ccgcaatgaa catgcagaaa catggtttct tgatgaaaac
     1081 cacccataca ggacatgggc ctaccacggg agctacgaag cccccacgca aggatcagcg
     1141 tcttccctcg tgaacggggt tgttagactc ctgtcaaagc cttgggacgt ggtgactgga
     1201 gttacaggaa tagccatgac tgacaccaca ccatacggcc aacaaagagt cttcaaagaa
     1261 aaagtggaca ccagggtgcc agatccccaa gaaggcactc gccaggtaat gaacatagtc
     1321 tcttcctggc tgtggaagga gctggggaaa cgcaagcggc cacgcgtctg caccaaagaa
     1381 gagtttatca acaaggtgcg cagcaatgca gcactgggag caatatttga agaggaaaaa
     1441 gaatggaaga cggctgtgga agctgtgaat gatccaaggt tttgggccct agtggatagg
     1501 gagagagaac accacctgag aggagagtgt cacagctgtg tgtacaacat gatgggaaaa
     1561 agagaaaaga agcaaggaga gttcgggaaa gcaaaaggta gccgcgccat ctggtacatg
     1621 tggttgggag ccagattctt ggagtttgaa gcccttggat tcttgaacga ggaccattgg
     1681 atgggaagag aaaactcagg aggtggagtc gaagggttag gattgcaaag acttggatac
     1741 attctagaag aaatgaatcg ggcaccagga ggaaagatgt acgcagatga cactgctggc
     1801 tgggacaccc gcattagtaa gtttgatctg gagaatgaag ctctgattac caaccaaatg
     1861 gaggaagggc acagaactct ggcgttggcc gtgattaaat acacatacca aaacaaagtg
     1921 gtgaaggttc tcagaccagc tgaaggagga aaaacagtta tggacatcat ttcaagacaa
     1981 gaccagagag ggagtggaca agttgtcact tatgctctca acacattcac caacttggtg
     2041 gtgcagctta tccggaacat ggaagctgag gaagtgttag agatgcaaga cttatggttg
     2101 ttgaggaagc cagagaaagt gaccagatgg ttgcagagca atggatggga tagactcaaa
     2161 cgaatggcgg tcagtggaga tgactgcgtt gtgaagccaa tcgatgatag gtttgcacat
     2221 gccctcaggt tcttgaatga catgggaaaa gttaggaaag acacacagga gtggaaaccc
     2281 tcgactggat ggagcaattg ggaagaagtc ccgttctgct cccaccactt caacaagctg
     2341 tacctcaagg atgggagatc cattgtggtc ccttgccgcc accaagatga actgattggc
     2401 cgagctcgcg tctcaccagg ggcaggatgg agcatccggg agactgcctg tcttgcaaaa
     2461 tcatatgcgc agatgtggca gctcctttat ttccacagaa gagatcttcg actgatggct
     2521 aatgccattt gctcggctgt gccagttgac tgggtaccaa ctgggagaac cacctggtca
     2581 atccacggaa agggagaatg gatgaccact gaggacatgc tcatggtgtg gaatagagtg
     2641 tggattgagg agaacgacca tatggaggac aagactcctg taacaaaatg gacagacatt
     2701 ccctatctag gaaaaaggga ggacttatgg tgtggatccc ttatagggca cagaccccgc
     2761 accacttggg ctgaaaacat caaagacaca gtcaacatgg tgcgcaggat cataggtgat
     2821 gaagaaaagt acatggacta tctatccacc caagtccgct acttgggtga ggaagggtcc
     2881 acacccggag tgttgaaccc agctttcttg tacaaagtgg tgctggaggt gctcttccag
     2941 ggcccgaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     3001 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     3061 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     3121 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     3181 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     3241 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     3301 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     3361 cagaagaaga ctatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     3421 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     3481 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     3541 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     3601 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagacctc tggtcaccac
     3661 catcaccacc atcaccacta agcggccgcc ctcctcctcc tttcttgttc ctttcacgtc
     3721 gccttctcgg ttgtagctgg cagacgacga gtcttacttt tacgtgtact tctctataga
     3781 tgatgtatga tctctctgca tgcgtgttcg tgcatgtgtc cgtgtgttgg gtacgcgtgg
     3841 taccctgcag gaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagtaatta
     3901 ctagcataac cccttggggc ctctaaacgg gtcttgaggg ggttttttgc tgaaaggagg
     3961 acagctgatg attgtcatgc ttgccatctg ttttcttgca aggtcagagg aattcgtaat
     4021 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4081 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4141 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4201 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4261 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4321 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4381 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4441 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4501 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4561 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4621 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4681 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4741 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4801 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4861 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4921 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4981 agcagcagat tacgcgcaga aaaaaaggat ctcaagagga tcctttgatc ttttctacgg
     5041 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5101 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5161 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5221 cgatctgtct agttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5281 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5341 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5401 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5461 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5521 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5581 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5641 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5701 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5761 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5821 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5881 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5941 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6001 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6061 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6121 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacct
//
