LOCUS       sequence-305165-        6717 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Cell-free/mammalian expression vector for Zika virus NS4Bprotein 
            with N-term mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6717)
  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 6717)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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_KA2_ZIKV NS4B
FEATURES             Location/Qualifiers
     source          1..6717
                     /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..3040
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="SKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQ
                     TAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFED
                     GGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGE
                     IKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEG
                     RHSTGGMDELYK"
     primer_bind     complement(2478..2496)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2746..2765
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3041..3064
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    3065..3089
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3846..3870)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3905..3922
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4012..4031)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4077..4132
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4131..4150)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4220..4267
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4280..4985
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4368..4740
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4397..4985)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4994..5027)
                     /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(5195..5782)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5274..5293)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5795..6586,6587..6655))
                     /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     6418..6437
                     /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 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     2401 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     2461 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     2521 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     2581 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     2641 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     2701 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     2761 gaagactatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     2821 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     2881 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     2941 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     3001 gggccgccac tccaccggcg gcatggacga gctgtacaag ctggaggtgc tcttccaggg
     3061 tccgacaagt ttgtacaaaa aagcaggctt caacgaactt ggatggctgg aaagaacaaa
     3121 aaatgacata gctcatctaa tgggaaggag agaagaagga gcaacgatgg gattctcaat
     3181 ggacattgat ctgcggccag cctccgcctg ggctatctat gccgcattga caactctcat
     3241 caccccagct gtccaacatg cggtaaccac ttcatacaac aactactcct taatggcgat
     3301 ggccacacaa gctggagtgc tgtttggcat gggcaaaggg atgccattta tgcatgggga
     3361 ccttggagtc ccgctgctaa tgatgggttg ctattcacaa ttaacacccc tgactctgat
     3421 agtagctatc attctgcttg tggcgcacta catgtacttg atcccaggcc tacaagcggc
     3481 agcagcgcgt gctgcccaga aaaggacagc agctggcatc atgaagaatc ccgttgtgga
     3541 tggaatagtg gtaactgaca ttgacacaat gacaatagac ccccaggtgg agaagaagat
     3601 gggacaagtg ttactcatag cagtagccat ctccagtgct gtgctgctgc ggaccgcctg
     3661 gggatggggg gaggctggag ctctgatcac agcagcgacc tccaccttgt gggaaggctc
     3721 tccaaacaaa tactggaact cctctacagc cacctcactg tgcaacatct tcagaggaag
     3781 ctatctggca ggagcttccc ttatctatac agtgacgaga aacgctggcc tggttaagag
     3841 acgtaaccca gctttcttgt acaaagtggt gtaagcggcc gccagctttc tagaccagtt
     3901 taaacatcac catcaccatc actaagtgat taacctcagg tgcaggctgc ctatcagaag
     3961 gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatcg atctttttcc
     4021 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata
     4081 aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag
     4141 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc
     4201 aacatatgcc catatgtaac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     4261 ttttttgctg aaagcatgcg gaggaaattc tccttgaagt ttccctggtg ttcaaagtaa
     4321 aggagtttgc accagacgca cctctgttca ctggtccggc gtattaaaac acgatacatt
     4381 gttattagta catttattaa gcgctagatt ctgtgcgttg ttgatttaca gacaattgtt
     4441 gtacgtattt taataattca ttaaatttat aatctttagg gtggtatgtt agagcgaaaa
     4501 tcaaatgatt ttcagcgtct ttatatctga atttaaatat taaatcctca atagatttgt
     4561 aaaataggtt tcgattagtt tcaaacaagg gttgtttttc cgaaccgatg gctggactat
     4621 ctaatggatt ttcgctcaac gccacaaaac ttgccaaatc ttgtagcagc aatctagctt
     4681 tgtcgatatt cgtttgtgtt ttgttttgta ataaaggttc gacgtcgttc aaaatattat
     4741 gcgcttttgt atttctttca tcactgtcgt tagtgtacaa ttgactcgac gtaaacacgt
     4801 taaatagagc ttggacatat ttaacatcgg gcgtgttagc tttattaggc cgattatcgt
     4861 cgtcgtccca accctcgtcg ttagaagttg cttccgaaga cgattttgcc atagccacac
     4921 gacgcctatt aattgtgtcg gctaacacgt ccgcgatcaa atttgtagtt gagctttttg
     4981 gaattgcgat cgcataactt cgtatagcat acattatacg aagttataag ctcggaacgc
     5041 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5101 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5161 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc cccctgacga
     5221 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5281 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5341 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5401 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5461 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5521 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5581 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5641 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5701 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5761 gcgcagaaaa aaaggatctc aagaagatcc tttgttacca atgcttaatc agtgaggcac
     5821 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5881 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5941 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6001 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6061 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6121 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6181 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6241 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6301 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6361 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6421 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6481 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6541 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6601 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6661 tcctttttca atattattga agcatttatc agggttattg tctcatgtcc gcgcgtt
//
