LOCUS       sequence-308746-        4967 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free expression vector for Zika virus Envelope protein with 
            C-term mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4967)
  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 4967)
  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 E
FEATURES             Location/Qualifiers
     source          1..4967
                     /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"
     CDS             1071..1082
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(1688..1712)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1714..1737
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1874..1892)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2142..2161
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2446..2469
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      2692..2738
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2816..2832)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2816..2832)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2829..2851)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2840..2856
                     /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(2864..2870,2871..2888,2889..2894))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2909..2930
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3047..3064)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3218..3806)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3298..3317)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3977..4768,4769..4837))
                     /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     4600..4619
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4838..4942)
                     /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 ggcttcatca ggtgcattgg agtcagcaat agagacttcg tggagggcat gtcaggtggg
      241 acctgggttg atgttgtctt ggaacatgga ggctgcgtta ccgtgatggc acaggacaag
      301 ccaacagtcg acatagagtt ggtcacgacg acggttagta acatggccga ggtaagatcc
      361 tattgctacg aggcatcgat atcggacatg gcttcggaca gtcgttgccc aacacaaggt
      421 gaagcctacc ttgacaagca atcagacact caatatgtct gcaaaagaac attagtggac
      481 agaggttggg gaaacggttg tggacttttt ggcaaaggga gcttggtgac atgtgccaag
      541 tttacgtgtt ctaagaagat gaccgggaag agcattcaac cggaaaatct ggagtatcgg
      601 ataatgctat cagtgcatgg ctcccagcat agcgggatga ttggatatga aactgacgaa
      661 gatagagcga aagtcgaggt tacgcctaat tcaccaagag cggaagcaac cttgggaggc
      721 tttggaagct taggacttga ctgtgaacca aggacaggcc ttgacttttc agatctgtat
      781 tacctgacca tgaacaataa gcattggttg gtgcacaaag agtggtttca tgacatccca
      841 ttgccttggc atgctggggc agacaccgga actccacact ggaacaacaa agaggcattg
      901 gtagaattca aggatgccca cgccaagagg caaaccgtcg tcgttctggg gagccaggaa
      961 ggagccgttc acacggctct cgctggagct ctagaggctg agatggatgg tgcaaaggga
     1021 aggctgttct ctggccattt gaaatgccgc ctaaaaatgg acaagcttag attgaagggc
     1081 gtgtcatatt ccttgtgcac tgcggcattc acattcacca aggtcccagc tgaaacactg
     1141 catggaacag tcacagtgga ggtgcagtat gcagggacag atggaccctg caagatccca
     1201 gtccagatgg cggtggacat gcagaccctg accccagttg gaaggctgat aaccgccaac
     1261 cccgtgatta ctgaaagcac tgagaactca aagatgatgt tggagcttga cccaccattt
     1321 ggggattctt acattgtcat aggagttggg gacaagaaaa tcacccacca ctggcatagg
     1381 agtggtagca ccatcggaaa ggcatttgag gccactgtga gaggcgccaa gagaatggca
     1441 gtcctggggg atacagcctg ggacttcgga tcagtcgggg gtgtgttcaa ctcactgggt
     1501 aagggcattc accagatttt tggagcagcc ttcaaatcac tgtttggagg aatgtcctgg
     1561 ttctcacaga tcctcatagg cacgctgcta gtgtggttag gtttgaacac aaagaatgga
     1621 tctatctccc tcacatgctt ggccctgggg ggagtgatga tcttcctctc cacggctgtt
     1681 tctgctaacc cagctttctt gtacaaagtg gtgctggagg tgctcttcca gggcccgaag
     1741 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     1801 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     1861 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     1921 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     1981 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     2041 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     2101 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
     2161 actatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     2221 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
     2281 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     2341 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     2401 cgccactcca ccggcggcat ggacgagctg tacaagacct ctggtcacca ccatcaccac
     2461 catcaccact aagcggccgc cctcctcctc ctttcttgtt cctttcacgt cgccttctcg
     2521 gttgtagctg gcagacgacg agtcttactt ttacgtgtac ttctctatag atgatgtatg
     2581 atctctctgc atgcgtgttc gtgcatgtgt ccgtgtgttg ggtacgcgtg gtaccctgca
     2641 ggaaggaagc tgagttggct gctgccaccg ctgagcaata actagtaatt actagcataa
     2701 ccccttgggg cctctaaacg ggtcttgagg gggttttttg ctgaaaggag gacagctgat
     2761 gattgtcatg cttgccatct gttttcttgc aaggtcagag gaattcgtaa tcatggtcat
     2821 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     2881 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     2941 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3001 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     3061 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3121 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3181 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3241 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3301 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3361 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3421 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3481 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3541 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3601 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3661 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3721 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3781 ttacgcgcag aaaaaaagga tctcaagagg atcctttgat cttttctacg gggtctgacg
     3841 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3901 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3961 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4021 tagttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4081 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4141 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4201 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4261 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     4321 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4381 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4441 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4501 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4561 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     4621 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4681 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4741 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4801 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4861 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4921 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacct
//
