LOCUS       sequence-305937-        7824 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/ mammalian expression vector for Zika virus NS3 protein 
            with N-term eGFP .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7824)
  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 7824)
  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: pmCellFree_KA1_ZIKV NS3
FEATURES             Location/Qualifiers
     source          1..7824
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             181..305
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(343..948)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1140..1443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1449..1726
                     /label=chicken beta-actin promoter
     primer_bind     2005..2027
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2089..2108
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2147..2166
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2147..2165
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2309..2332
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2339..3049
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             2339..3049
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(2639..2658)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2986..3007
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3050..3073
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    3074..3098
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4953..4977)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5012..5029
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5119..5138)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5184..5239
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5238..5257)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      5327..5374
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     5387..6092
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           5475..5847
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(5504..6092)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(6101..6134)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(6302..6889)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6381..6400)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6902..7693,7694..7762))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7525..7544
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga
       61 ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat
      121 gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca
      181 tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa
      241 ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt
      301 attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa
      361 cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag
      421 gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt
      481 ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt
      541 ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt
      601 aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg
      661 catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc
      721 tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt
      781 gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt
      841 aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc
      901 tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg
      961 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggccg
     1021 taatgagacg cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctct
     1081 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     1141 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     1201 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     1261 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     1321 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     1381 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     1441 atgcatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     1501 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
     1561 gggggggggg cgcgcgccgg gggggggggg gggggggggg gggggggggg ggggggggcg
     1621 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
     1681 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
     1741 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     1801 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctccttcgg gctgtaatta
     1861 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
     1921 ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc
     1981 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag
     2041 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg
     2101 ttattgtgct gtctcatcat tttggcaaag aattggatcg gacgtctaat acgactcact
     2161 atagggacat cttaagttta ttttatttta ttttatttta ttttatttta ttttatttta
     2221 ttttatttta ttttatttaa ccatgacagt aatgtataaa gtctgtaaag acattaaaca
     2281 cgtaagtgaa accatgggca cctctggtca ccaccatcac caccatcacc acggcacaag
     2341 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagttgg acggcgacgt
     2401 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     2461 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2521 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     2581 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2641 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2701 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     2761 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     2821 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     2881 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     2941 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     3001 gttcgtgacc gccgccggga tcactctcgg catggacgag ctatacaagc tggaggtgct
     3061 cttccagggt ccgacaagtt tgtacaaaaa agcaggcttc agtggcgccc tctgggacgt
     3121 gcctgctccc aaagaagtga agaaaggaga gaccacagat ggagtgtaca gagtgatgac
     3181 tcgcagactg ctaggttcaa cacaggttgg agtgggagtc atgcaagagg gagtcttcca
     3241 caccatgtgg cacgttacaa aaggagccgc actgaggagc ggtgagggaa gacttgatcc
     3301 atactggggg gatgtcaagc aggacttggt gtcatactgt gggccttgga agttggatgc
     3361 agcttgggat ggactcagcg aggtacagct tttggccgta cctcccggag agagggccag
     3421 aaacattcag accctgcctg gaatattcaa gacaaaggac ggggacatcg gagcagttgc
     3481 tctggactac cctgcaggga cctcaggatc tccgatccta gacaaatgtg gaagagtgat
     3541 aggactctat ggcaatgggg ttgtgatcaa gaatggaagc tatgttagtg ctataaccca
     3601 gggaaagagg gaggaggaga ctccggttga atgtttcgaa ccctcgatgc tgaagaagaa
     3661 gcagctaact gtcttggatc tgcatccagg agccggaaaa accaggagag ttcttcctga
     3721 aatagtccgt gaagccataa aaaagagact ccggacagtg atcttggcac caactagggt
     3781 tgtcgctgct gagatggagg aggccttgag aggacttccg gtgcgttaca tgacaacagc
     3841 agtcaacgtc acccattctg ggacagaaat cgttgatttg atgtgccatg ccactttcac
     3901 ttcacgctta ctacaaccca tcagagtccc taattacaat ctcaacatca tggatgaagc
     3961 ccacttcaca gacccctcaa gtatagctgc aagaggatac atatcaacaa gggttgaaat
     4021 gggcgaggcg gctgccattt ttatgactgc cacaccacca ggaacccgtg atgcgtttcc
     4081 tgactctaac tcaccaatca tggacacaga agtggaagtc ccagagagag cctggagctc
     4141 aggctttgat tgggtgacag accattctgg gaaaacagtt tggttcgttc caagcgtgag
     4201 aaacggaaat gaaatcgcag cctgtctgac aaaggctgga aagcgggtca tacagctcag
     4261 caggaagact tttgagacag aatttcagaa aacaaaaaat caagagtggg actttgtcat
     4321 aacaactgac atctcagaga tgggcgccaa cttcaaggct gaccgggtca tagactctag
     4381 gagatgccta aaaccagtca tacttgatgg tgagagagtc atcttggctg ggcccatgcc
     4441 tgtcacgcat gctagtgctg ctcagaggag aggacgtata ggcaggaacc ctaacaaacc
     4501 tggagatgag tacatgtatg gaggtgggtg tgcagagact gatgaaggcc atgcacactg
     4561 gcttgaagca agaatgcttc ttgacaacat ctacctccag gatggcctca tagcctcgct
     4621 ctatcggcct gaggccgata aggtagccgc cattgaggga gagtttaagc tgaggacaga
     4681 gcaaaggaag accttcgtgg aactcatgaa gagaggtgac cttcccgtct ggctagccta
     4741 tcaggttgca tctgccggaa taacttacac agacagaaga tggtgctttg atggcacaac
     4801 caacaacacc ataatggaag acagtgtacc agcagaggtt tggacaaagt atggagagaa
     4861 gagagtgctc aaaccgagat ggatggatgc tagggtctgt tcagaccatg cggccctgaa
     4921 gtcgttcaaa gaattcgccg ctggaaaaag aaacccagct ttcttgtaca aagtggtgta
     4981 agcggccgcc agctttctag accagtttaa acatcaccat caccatcact aagtgattaa
     5041 cctcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc cctggctcac
     5101 aaataccact gagatcgatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc
     5161 ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg
     5221 gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca
     5281 gaatgagtat ttggtttaga gtttggcaac atatgcccat atgtaactag cataacccct
     5341 tggggcctct aaacgggtct tgaggggttt tttgctgaaa gcatgcggag gaaattctcc
     5401 ttgaagtttc cctggtgttc aaagtaaagg agtttgcacc agacgcacct ctgttcactg
     5461 gtccggcgta ttaaaacacg atacattgtt attagtacat ttattaagcg ctagattctg
     5521 tgcgttgttg atttacagac aattgttgta cgtattttaa taattcatta aatttataat
     5581 ctttagggtg gtatgttaga gcgaaaatca aatgattttc agcgtcttta tatctgaatt
     5641 taaatattaa atcctcaata gatttgtaaa ataggtttcg attagtttca aacaagggtt
     5701 gtttttccga accgatggct ggactatcta atggattttc gctcaacgcc acaaaacttg
     5761 ccaaatcttg tagcagcaat ctagctttgt cgatattcgt ttgtgttttg ttttgtaata
     5821 aaggttcgac gtcgttcaaa atattatgcg cttttgtatt tctttcatca ctgtcgttag
     5881 tgtacaattg actcgacgta aacacgttaa atagagcttg gacatattta acatcgggcg
     5941 tgttagcttt attaggccga ttatcgtcgt cgtcccaacc ctcgtcgtta gaagttgctt
     6001 ccgaagacga ttttgccata gccacacgac gcctattaat tgtgtcggct aacacgtccg
     6061 cgatcaaatt tgtagttgag ctttttggaa ttgcgatcgc ataacttcgt atagcataca
     6121 ttatacgaag ttataagctc ggaacgctgc gctcggtcgt tcggctgcgg cgagcggtat
     6181 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     6241 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     6301 ttttccatag gctccgcccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6361 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6421 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6481 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6541 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6601 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6661 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6721 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6781 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6841 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6901 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6961 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     7021 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     7081 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     7141 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     7201 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     7261 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     7321 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     7381 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     7441 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     7501 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     7561 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     7621 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     7681 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     7741 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7801 gttattgtct catgtccgcg cgtt
//
