LOCUS       sequence-304982-        7473 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for Zika virus Envelope 
            protein with N-term eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7473)
  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 7473)
  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 E
FEATURES             Location/Qualifiers
     source          1..7473
                     /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"
     CDS             3985..3996
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(4602..4626)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4661..4678
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4768..4787)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4833..4888
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4887..4906)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4976..5023
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     5036..5741
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           5124..5496
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(5153..5741)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5750..5783)
                     /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(5951..6538)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6030..6049)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6551..7342,7343..7411))
                     /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     7174..7193
                     /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 atcaggtgca ttggagtcag
     3121 caatagagac ttcgtggagg gcatgtcagg tgggacctgg gttgatgttg tcttggaaca
     3181 tggaggctgc gttaccgtga tggcacagga caagccaaca gtcgacatag agttggtcac
     3241 gacgacggtt agtaacatgg ccgaggtaag atcctattgc tacgaggcat cgatatcgga
     3301 catggcttcg gacagtcgtt gcccaacaca aggtgaagcc taccttgaca agcaatcaga
     3361 cactcaatat gtctgcaaaa gaacattagt ggacagaggt tggggaaacg gttgtggact
     3421 ttttggcaaa gggagcttgg tgacatgtgc caagtttacg tgttctaaga agatgaccgg
     3481 gaagagcatt caaccggaaa atctggagta tcggataatg ctatcagtgc atggctccca
     3541 gcatagcggg atgattggat atgaaactga cgaagataga gcgaaagtcg aggttacgcc
     3601 taattcacca agagcggaag caaccttggg aggctttgga agcttaggac ttgactgtga
     3661 accaaggaca ggccttgact tttcagatct gtattacctg accatgaaca ataagcattg
     3721 gttggtgcac aaagagtggt ttcatgacat cccattgcct tggcatgctg gggcagacac
     3781 cggaactcca cactggaaca acaaagaggc attggtagaa ttcaaggatg cccacgccaa
     3841 gaggcaaacc gtcgtcgttc tggggagcca ggaaggagcc gttcacacgg ctctcgctgg
     3901 agctctagag gctgagatgg atggtgcaaa gggaaggctg ttctctggcc atttgaaatg
     3961 ccgcctaaaa atggacaagc ttagattgaa gggcgtgtca tattccttgt gcactgcggc
     4021 attcacattc accaaggtcc cagctgaaac actgcatgga acagtcacag tggaggtgca
     4081 gtatgcaggg acagatggac cctgcaagat cccagtccag atggcggtgg acatgcagac
     4141 cctgacccca gttggaaggc tgataaccgc caaccccgtg attactgaaa gcactgagaa
     4201 ctcaaagatg atgttggagc ttgacccacc atttggggat tcttacattg tcataggagt
     4261 tggggacaag aaaatcaccc accactggca taggagtggt agcaccatcg gaaaggcatt
     4321 tgaggccact gtgagaggcg ccaagagaat ggcagtcctg ggggatacag cctgggactt
     4381 cggatcagtc gggggtgtgt tcaactcact gggtaagggc attcaccaga tttttggagc
     4441 agccttcaaa tcactgtttg gaggaatgtc ctggttctca cagatcctca taggcacgct
     4501 gctagtgtgg ttaggtttga acacaaagaa tggatctatc tccctcacat gcttggccct
     4561 ggggggagtg atgatcttcc tctccacggc tgtttctgct aacccagctt tcttgtacaa
     4621 agtggtgtaa gcggccgcca gctttctaga ccagtttaaa catcaccatc accatcacta
     4681 agtgattaac ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc
     4741 ctggctcaca aataccactg agatcgatct ttttccctct gccaaaaatt atggggacat
     4801 catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat
     4861 agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt
     4921 aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata tgtaactagc
     4981 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag catgcggagg
     5041 aaattctcct tgaagtttcc ctggtgttca aagtaaagga gtttgcacca gacgcacctc
     5101 tgttcactgg tccggcgtat taaaacacga tacattgtta ttagtacatt tattaagcgc
     5161 tagattctgt gcgttgttga tttacagaca attgttgtac gtattttaat aattcattaa
     5221 atttataatc tttagggtgg tatgttagag cgaaaatcaa atgattttca gcgtctttat
     5281 atctgaattt aaatattaaa tcctcaatag atttgtaaaa taggtttcga ttagtttcaa
     5341 acaagggttg tttttccgaa ccgatggctg gactatctaa tggattttcg ctcaacgcca
     5401 caaaacttgc caaatcttgt agcagcaatc tagctttgtc gatattcgtt tgtgttttgt
     5461 tttgtaataa aggttcgacg tcgttcaaaa tattatgcgc ttttgtattt ctttcatcac
     5521 tgtcgttagt gtacaattga ctcgacgtaa acacgttaaa tagagcttgg acatatttaa
     5581 catcgggcgt gttagcttta ttaggccgat tatcgtcgtc gtcccaaccc tcgtcgttag
     5641 aagttgcttc cgaagacgat tttgccatag ccacacgacg cctattaatt gtgtcggcta
     5701 acacgtccgc gatcaaattt gtagttgagc tttttggaat tgcgatcgca taacttcgta
     5761 tagcatacat tatacgaagt tataagctcg gaacgctgcg ctcggtcgtt cggctgcggc
     5821 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5881 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5941 tgctggcgtt tttccatagg ctccgccccc tgacgagcat cacaaaaatc gacgctcaag
     6001 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     6061 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     6121 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     6181 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     6241 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6301 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     6361 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     6421 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6481 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     6541 agatcctttg ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6601 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6661 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6721 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6781 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6841 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6901 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6961 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7021 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7081 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     7141 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     7201 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7261 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7321 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7381 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7441 tttatcaggg ttattgtctc atgtccgcgc gtt
//
