LOCUS       sequence-305785-        6362 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/ mammalian expression vector for Zika virus NS2B protein 
            with N-term eGFP .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6362)
  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 6362)
  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 NS2B
FEATURES             Location/Qualifiers
     source          1..6362
                     /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(3491..3515)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3550..3567
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3657..3676)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3722..3777
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3776..3795)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3865..3912
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3925..4630
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4013..4385
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4042..4630)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4639..4672)
                     /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(4840..5427)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4919..4938)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5440..6231,6232..6300))
                     /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     6063..6082
                     /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 gcaggcttca gctggccccc tagtgaagtt
     3121 ctcacagccg ttggcctgat atgtgcactg gccggagggt ttgccaaggc agacattgag
     3181 atggctggac cgatggctgc agtaggcttg ctaattgtca gctatgtggt ctcgggaaag
     3241 agtgtggaca tgtacattga aagagcagga gacatcacat gggaaaagga cgcggaagtc
     3301 actggaaaca gtcctcggct tgacgtggca ctggatgaga gtggtgactt ctccttggta
     3361 gaggaagatg gtccacccat gagagagatc atactcaagg tggtcctgat ggccatctgt
     3421 ggcatgaacc caatagctat accttttgct gcaggagcgt ggtatgtgta tgtgaagact
     3481 gggaaaagga acccagcttt cttgtacaaa gtggtgtaag cggccgccag ctttctagac
     3541 cagtttaaac atcaccatca ccatcactaa gtgattaacc tcaggtgcag gctgcctatc
     3601 agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga gatcgatctt
     3661 tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc
     3721 taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc
     3781 ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt
     3841 ttggcaacat atgcccatat gtaactagca taaccccttg gggcctctaa acgggtcttg
     3901 aggggttttt tgctgaaagc atgcggagga aattctcctt gaagtttccc tggtgttcaa
     3961 agtaaaggag tttgcaccag acgcacctct gttcactggt ccggcgtatt aaaacacgat
     4021 acattgttat tagtacattt attaagcgct agattctgtg cgttgttgat ttacagacaa
     4081 ttgttgtacg tattttaata attcattaaa tttataatct ttagggtggt atgttagagc
     4141 gaaaatcaaa tgattttcag cgtctttata tctgaattta aatattaaat cctcaataga
     4201 tttgtaaaat aggtttcgat tagtttcaaa caagggttgt ttttccgaac cgatggctgg
     4261 actatctaat ggattttcgc tcaacgccac aaaacttgcc aaatcttgta gcagcaatct
     4321 agctttgtcg atattcgttt gtgttttgtt ttgtaataaa ggttcgacgt cgttcaaaat
     4381 attatgcgct tttgtatttc tttcatcact gtcgttagtg tacaattgac tcgacgtaaa
     4441 cacgttaaat agagcttgga catatttaac atcgggcgtg ttagctttat taggccgatt
     4501 atcgtcgtcg tcccaaccct cgtcgttaga agttgcttcc gaagacgatt ttgccatagc
     4561 cacacgacgc ctattaattg tgtcggctaa cacgtccgcg atcaaatttg tagttgagct
     4621 ttttggaatt gcgatcgcat aacttcgtat agcatacatt atacgaagtt ataagctcgg
     4681 aacgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     4741 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     4801 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgccccct
     4861 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4921 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4981 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5041 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5101 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5161 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5221 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5281 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5341 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5401 attacgcgca gaaaaaaagg atctcaagaa gatcctttgt taccaatgct taatcagtga
     5461 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5521 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5581 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5641 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     5701 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     5761 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     5821 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     5881 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     5941 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6001 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     6061 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     6121 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6181 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6241 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6301 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgtccgcgcg
     6361 tt
//
