LOCUS       sequence-304984-        7028 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for Zika virus NS1 protein 
            with N-term eGFP .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7028)
  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 7028)
  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 NS1
FEATURES             Location/Qualifiers
     source          1..7028
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     180..1007
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             180..304
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(342..947)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1139..1442
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1448..1725
                     /label=chicken beta-actin promoter
     primer_bind     2004..2026
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2088..2107
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2146..2165
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2146..2164
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2308..2331
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2338..3048
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             2338..3048
                     /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(2638..2657)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2985..3006
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3049..3072
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    3073..3097
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4157..4181)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4216..4233
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4323..4342)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4388..4443
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4442..4461)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4531..4578
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4591..5296
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4679..5051
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4708..5296)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5305..5338)
                     /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(5506..6093)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5585..5604)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6106..6897,6898..6966))
                     /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     6729..6748
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cctgcatctt ttaatcaaat cccaagatgt gtataaacgc gccggtatgt acaggaagag
       61 gtttatacta aactgttaca ttgcaaacgt ggtttcgtgt gccaagtgtg aaaaccgatg
      121 tttaatcaag gctctgacgc atttctacaa ccacgactcc aagtgtgtgg gtgaagtcat
      181 gcatctttta atcaaatccc aagatgtgta taaaccacca aactgccaaa aaatgaaaac
      241 tgtcgacaag ctctgtccgt ttgctggcaa ctgcaagggt ctcaatccta tttgtaatta
      301 ttgaataata aaacaattat aaatgtcaaa tttgtttttt attaacgata caaaccaaac
      361 gcaacaagaa catttgtagt attatctata attgaaaacg cgtagttata atcgctgagg
      421 taatatttaa aatcattttc aaatgattca cagttaattt gcgacaatat aattttattt
      481 tcacataaac tagacgcctt gtcgtcttct tcttcgtatt ccttctcttt ttcatttttc
      541 tcttcataaa aattaacata gttattatcg tatccatata tgtatctatc gtatagagta
      601 aattttttgt tgtcataaat atatatgtct tttttaatgg ggtgtatagt accgctgcgc
      661 atagtttttc tgtaatttac aacagtgcta ttttctggta gttcttcgga gtgtgttgct
      721 ttaattatta aatttatata atcaatgaat ttgggatcgt cggttttgta caatatgttg
      781 ccggcatagt acgcagcttc ttctagttca attacaccat tttttagcag caccggatta
      841 acataacttt ccaaaatgtt gtacgaaccg ttaaacaaaa acagttcacc tcccttttct
      901 atactattgt ctgcgagcag ttgtttgttg ttaaaaataa cagccattgt aatgagacgc
      961 acaaactaat atcacaaact ggaaatgtct atcaatatat agttgctgat ggccggccgt
     1021 aatgagacgc acaaactaat atcacaaact ggaaatgtct atcaatatat agttgctcta
     1081 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     1141 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     1201 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     1261 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     1321 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     1381 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     1441 tgcatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca
     1501 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
     1561 gggggggggc gcgcgccggg gggggggggg gggggggggg gggggggggg gggggggcgg
     1621 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
     1681 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
     1741 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     1801 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctccttcggg ctgtaattag
     1861 cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc
     1921 cgggagggcc ctttgtgcgg ggggagcggc tcggggctgt ccgcgggggg acggctgcct
     1981 tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc
     2041 ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt
     2101 tattgtgctg tctcatcatt ttggcaaaga attggatcgg acgtctaata cgactcacta
     2161 tagggacatc ttaagtttat tttattttat tttattttat tttattttat tttattttat
     2221 tttattttat tttatttaac catgacagta atgtataaag tctgtaaaga cattaaacac
     2281 gtaagtgaaa ccatgggcac ctctggtcac caccatcacc accatcacca cggcacaagc
     2341 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagttgga cggcgacgta
     2401 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     2461 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     2521 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     2581 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     2641 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     2701 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     2761 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     2821 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     2881 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2941 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     3001 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tatacaagct ggaggtgctc
     3061 ttccagggtc cgacaagttt gtacaaaaaa gcaggcttcg acgtggggtg ctcagtggac
     3121 ttctcaaaaa aggaaacgag atgtggcacg ggggtattca tctataatga tgttgaagcc
     3181 tggagggacc ggtacaagta ccatcctgac tccccccgca gattggcagc agcagtcaag
     3241 caggcctggg aagaggggat ctgtgggatc tcatccgttt caagaatgga aaacatcatg
     3301 tggaaatcag tagaagggga gctcaatgct atcctagagg agaatggagt tcaactgaca
     3361 gttgttgtgg gatctgtaaa aaaccccatg tggagaggtc cacaaagatt gccagtgcct
     3421 gtgaatgagc tgccccacgg ctggaaagcc tgggggaaat cgtattttgt tagggcggca
     3481 aagaccaaca acagttttgt tgtcgacggt gacacactga aggaatgtcc gcttgagcac
     3541 agagcatgga atagttttct tgtggaggat cacgggtttg gagtcttcca caccagtgtc
     3601 tggcttaagg tcagagaaga ttactcatta gaatgtgacc cagccgtcat aggaacagct
     3661 gttaagggaa gggaggccgc gcacagtgat ctgggctatt ggattgaaag tgaaaagaat
     3721 gacacatgga ggctgaagag ggcccacctg attgagatga aaacatgtga atggccaaag
     3781 tctcacacat tgtggacaga tggagtagaa gaaagtgatc ttatcatacc caagtcttta
     3841 gctggtccac tcagccacca caacaccaga gagggttaca gaacccaagt gaaagggcct
     3901 tggcacagtg aagagcttga aatccggttt gaggaatgtc caggcaccaa ggtttacgtg
     3961 gaggagacat gcggaactag aggaccatct ctgagatcaa ctactgcaag tggaagggtc
     4021 attgaggaat ggtgctgtag ggaatgcaca atgcccccac tatcgtttcg agcaaaagac
     4081 ggctgctggt atggaatgga gataaggccc aggaaagaac cagagagcaa cttagtgagg
     4141 tcaatggtga cagcgaaccc agctttcttg tacaaagtgg tgtaagcggc cgccagcttt
     4201 ctagaccagt ttaaacatca ccatcaccat cactaagtga ttaacctcag gtgcaggctg
     4261 cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc
     4321 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
     4381 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
     4441 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt
     4501 tagagtttgg caacatatgc ccatatgtaa ctagcataac cccttggggc ctctaaacgg
     4561 gtcttgaggg gttttttgct gaaagcatgc ggaggaaatt ctccttgaag tttccctggt
     4621 gttcaaagta aaggagtttg caccagacgc acctctgttc actggtccgg cgtattaaaa
     4681 cacgatacat tgttattagt acatttatta agcgctagat tctgtgcgtt gttgatttac
     4741 agacaattgt tgtacgtatt ttaataattc attaaattta taatctttag ggtggtatgt
     4801 tagagcgaaa atcaaatgat tttcagcgtc tttatatctg aatttaaata ttaaatcctc
     4861 aatagatttg taaaataggt ttcgattagt ttcaaacaag ggttgttttt ccgaaccgat
     4921 ggctggacta tctaatggat tttcgctcaa cgccacaaaa cttgccaaat cttgtagcag
     4981 caatctagct ttgtcgatat tcgtttgtgt tttgttttgt aataaaggtt cgacgtcgtt
     5041 caaaatatta tgcgcttttg tatttctttc atcactgtcg ttagtgtaca attgactcga
     5101 cgtaaacacg ttaaatagag cttggacata tttaacatcg ggcgtgttag ctttattagg
     5161 ccgattatcg tcgtcgtccc aaccctcgtc gttagaagtt gcttccgaag acgattttgc
     5221 catagccaca cgacgcctat taattgtgtc ggctaacacg tccgcgatca aatttgtagt
     5281 tgagcttttt ggaattgcga tcgcataact tcgtatagca tacattatac gaagttataa
     5341 gctcggaacg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5401 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5461 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5521 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5581 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5641 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5701 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5761 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5821 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5881 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5941 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6001 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     6061 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgttacc aatgcttaat
     6121 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6181 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6241 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6301 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6361 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6421 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6481 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6541 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6601 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6661 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6721 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6781 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6841 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6901 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6961 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgtc
     7021 cgcgcgtt
//
