LOCUS       sequence-496342-        5391 bp    DNA     circular SYN 07-JAN-2026
DEFINITION  T7-driven EBOV minigenome vRNA with Zeo-eGFP reporter selection 
            cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5391)
  TITLE     bioRxiv preprint: Genome-wide CRISPR knockout screening with viral 
            replicons for identification of host factors involved in viral 
            replication
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5391)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 7, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: EBOV Zeo-eGFP minigenome
FEATURES             Location/Qualifiers
     source          1..5391
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        486..504
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(1240..1953,1954..1956,1957..1959))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1243..1953,1954..1956,1957..1959))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1285..1306)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1634..1653
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1893..1914
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2961..2980)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3026..3081
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3080..3099)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3170..3186)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3170..3186)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3183..3205)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3194..3210
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3218..3224,3225..3242,3243..3248))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3263..3284
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3401..3418)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3572..4160)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3652..3671)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4331..5122,5123..5191))
                     /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     4954..4973
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5192..5296)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5364..5382
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa
      421 tgcatctaga tggtctcata tgctcgaggg tacccccggg gagctcttaa ttaatcgatg
      481 ctagctaata cgactcacta taggacacac aaaaaagaag aaatagattt atttttaaat
      541 ttttgtgtgc gaccattttt ccaggaatcc tttttgcaac gtttattctg gcgagccttc
      601 tgatgagcgt ggtcaatgtc ttaattatca ttaacacgaa gattattatc tagtctgcta
      661 tattgtaaag aaggtatttc tgattttaca gtcctgcctt ttcttttaat tttatcattc
      721 tgtatgaaat agtagcttac tagacaaaaa tgttaaggtt attacaccta atctcttatt
      781 ttacacaaag attaaggcta tcaccgtttt taaaacgaca aattaaagta atgataatta
      841 taaaaaactt aatcttaaag atcaattttg ttttaagaag ttgtgttatc aaccaaaaca
      901 ctattccatc tgacaggata ttgatacaac attattatat caataacaat atgagcccag
      961 acctttcgtt aaagcttaat tataaagagt gcagagttta ttatgttgcg ttaaattcat
     1021 tgcggcagta atatgtcaca catgctgcat tgtgaatttt ttgattatca cgcagggaga
     1081 gaggctaaat atagtatata atatagggac tcctttcgtt atttaaatag ttgtttattg
     1141 agattagatc aatatgatgt atgagagcaa tttatgagtt ttttataatc tgtatgttaa
     1201 taaccaacct ttgcaagtat caggatacta tgcggccgct cacttgtaca gctcgtccat
     1261 gccgagagtg atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc
     1321 gttggggtct ttgctcaggg cggactgggt gctcaggtag tggttgtcgg gcagcagcac
     1381 ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc
     1441 ctcgatgttg tggcggatct tgaagttcac cttgatgccg ttcttctgct tgtcggccat
     1501 gatatagacg ttgtggctgt tgtagttgta ctccagcttg tgccccagga tgttgccgtc
     1561 ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt
     1621 cacctcggcg cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac
     1681 gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg
     1741 gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg gcacgggcag
     1801 cttgccggtg gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc
     1861 ctcgccggac acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg
     1921 caccaccccg gtgaacagct cctcgccctt gctcaccatg ctgccgctac cgctaccgct
     1981 gccgctacca tcttgttctt cagcaacaaa atgaacacaa tttccagctg gatctctcaa
     2041 agcaaattct ctaccccatg gctgttcacc aatctcggtc atagctggac cagaagcatc
     2101 tctgaagttt gtagatacaa cttctgacca ttcagcatat aattcatcta aacccctgac
     2161 ccagacccaa gccaatgtat tatcagggac aacttgatct tggactgctg agataaataa
     2221 agtaacgtca tccctgacaa caccagcaaa atcgtcctca acaaagtctc ttgagaatcc
     2281 taacctatct gtccaaaact ctacagcacc tgcaacatcc ctagctgtta aaacaggaac
     2341 ggcagatgtt aatttagcca tcatacgcgt atgagacata ctcggaattt tgtgattccg
     2401 agcaatttgt cttttaacta tttccaatgt tttaccccaa gctttagggt tgttgaatct
     2461 tcaatcccct aagtgttcaa gttcaaaacc aaggttaatt ctcaatcttg cttgtttgac
     2521 tgtgaactaa tgctgtcctt taacaataat gtttctatta aggttgttgg atcaccctgc
     2581 agagtctgag gtggaacttg tgatgtgata agacctaggt gttgacaagg gattttcaac
     2641 tgagcacact gatgcccttg ctcccttcgg agacctaggt tgttctctat ctttgtgatg
     2701 tggctctgaa acaaaccagg tgtgattaca gtaacaattt caatttaaat tccgatataa
     2761 atttcaatga gaggaaaatt attaatcttc ctcatagtta ttcgcacaca aaagatccta
     2821 aaaattcttc tttctttttg tgtgtccggg gtcggcatgg catctccacc tcctcgcggt
     2881 ccgacctggg catccgaagg aggacgtcgt ccactcggat ggctaaggga gagctcccta
     2941 gatcggaact cgctagcaga tctttttccc tctgccaaaa attatgggga catcatgaag
     3001 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     3061 tggaattttt tgtgtctctc actcggaagg acatatgggg gatcctgaga ccatcggatc
     3121 ccgggcccgt cgactgcaga ggcctgcatg caagcttggc gtaatcatgg tcatagctgt
     3181 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     3241 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     3301 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     3361 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     3421 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3481 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3541 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3601 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3661 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3721 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3781 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3841 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3901 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3961 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4021 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4081 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4141 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4201 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4261 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4321 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4381 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     4441 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     4501 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4561 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4621 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4681 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4741 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4801 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4861 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4921 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4981 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5041 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5101 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5161 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5221 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5281 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     5341 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt c
//
