LOCUS       sequence-404987-        5993 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Co-express EGFP and SARS-CoV-2 ORF6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5993)
  AUTHORS   Yoo TY, Mitchison TJ
  TITLE     Quantitative comparison of nuclear transport inhibition by SARS 
            coronavirus ORF6 reveals the importance of oligomerization.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Jan 23;121(4):e2307997121. doi: 
            10.1073/pnas.2307997121. Epub 2024 Jan 18.
  PUBMED    38236733
REFERENCE   2  (bases 1 to 5993)
  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"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2024-01-23"; volume: "121(4):e2307997121. 
            doi"; pages: " 10.1073/pnas.2307997121. Epub 2024 Jan 18"
COMMENT     Sequence Label: pTY153 EGFP-T2A-ORF6 SARS-CoV-2
FEATURES             Location/Qualifiers
     source          1..5993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..588
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     539..559
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     633..652
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        633..651
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      672..681
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(678..680,681..683,684..1394)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(678..680,681..683,684..1394)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(723..744)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(984..1003)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1331..1352
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1404..1457
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(1771..1788)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1777..2001
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2047..2475
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2134..2153)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2344..2365
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2484..2504)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2489..2818
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2669..2804
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2731..2750
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2866..2913
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2932..3306
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    3436..3557
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3473..3492)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3527..3546
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3606..3622)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3606..3622)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3619..3641)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3630..3646
                     /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(3654..3660,3661..3678,3679..3684))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3837..3854)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4008..4596)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4088..4107)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4767..5558,5559..5627))
                     /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     5390..5409
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5628..5732)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5807..5826)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      421 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      481 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      541 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      601 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
      661 gctggctagt cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      721 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
      781 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
      841 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
      901 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
      961 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1021 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1081 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     1141 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     1201 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1261 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     1321 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     1381 acgagctgta caagggcagt ggagagggca gaggaagtct gctaacatgc ggtgacgtcg
     1441 aggagaatcc tggcccaatg ttccacctcg tagatttcca ggtgacgatc gccgagatct
     1501 tgctcataat aatgcggacc ttcaaggtta gcatttggaa tctcgattat ataattaact
     1561 taataattaa aaatctgagc aagtcactga ctgaaaataa atacagccaa ctggacgaag
     1621 aacagccgat ggagatcgat tgatgaagag gacaacatgg ccagtctgcc tgccacacat
     1681 gagctccaca tatttgggtc cataaatgga gtagatttcg atatggtagg acaagggact
     1741 ggcaacccga atgacggacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     1801 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     1861 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     1921 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     1981 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     2041 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2101 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2161 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2221 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2281 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2341 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2401 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     2461 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     2521 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     2581 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2641 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     2701 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     2761 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     2821 gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca
     2881 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag
     2941 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg
     3001 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg
     3061 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg
     3121 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc
     3181 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg
     3241 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag
     3301 gactgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     3361 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     3421 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3481 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3541 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     3601 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3661 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta acttacatta
     3721 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3781 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3841 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3901 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3961 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4021 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4081 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4141 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4201 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4261 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4321 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4381 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4441 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4501 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4561 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4621 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4681 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4741 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4801 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4861 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4921 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4981 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5041 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5101 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5161 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5221 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5281 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5341 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5401 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5461 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5521 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5581 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5641 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5701 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     5761 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     5821 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     5881 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     5941 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acg
//
