LOCUS       sequence-404985-        6044 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-express EGFP and SARS-CoV-2 ORF6 N-terminally labeled with ALFA 
            tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6044)
  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 6044)
  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"; 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: pTY186 EGFP-T2A-ALFA-ORF6 SARS-CoV-2
FEATURES             Location/Qualifiers
     source          1..6044
                     /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"
     CDS             1461..1499
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     primer_bind     complement(1822..1839)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1828..2052
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2098..2526
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2185..2204)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2395..2416
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2535..2555)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2540..2869
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2720..2855
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2782..2801
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2917..2964
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2983..3357
                     /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    3487..3608
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3524..3543)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3578..3597
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3657..3673)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3657..3673)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3670..3692)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3681..3697
                     /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(3705..3711,3712..3729,3730..3735))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3888..3905)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4059..4647)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4139..4158)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4818..5609,5610..5678))
                     /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     5441..5460
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5679..5783)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5858..5877)
                     /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 tggcccacca tcccgactcg aagaagagct gaggcggagg cttacggaac
     1501 ctgggtcaat gttccacctc gtagatttcc aggtgacgat cgccgagatc ttgctcataa
     1561 taatgcggac cttcaaggtt agcatttgga atctcgatta tataattaac ttaataatta
     1621 aaaatctgag caagtcactg actgaaaata aatacagcca actggacgaa gaacagccga
     1681 tggagatcga ttgatgaaga ggacaacatg gccagtctgc ctgccacaca tgagctccac
     1741 atatttgggt ccataaatgg agtagatttc gatatggtag gacaagggac tggcaacccg
     1801 aatgacggac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     1861 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     1921 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     1981 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     2041 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     2101 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2161 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2221 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2281 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2341 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2401 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     2461 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     2521 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     2581 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     2641 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     2701 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     2761 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     2821 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     2881 agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag
     2941 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt
     3001 gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg
     3061 ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg
     3121 accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg
     3181 tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc
     3241 cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc
     3301 gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac
     3361 gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     3421 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3481 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3541 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3601 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     3661 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3721 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aacttacatt aattgcgttg
     3781 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3841 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3901 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3961 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4021 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4081 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4141 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4201 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4261 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4321 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4381 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4441 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4501 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4561 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4621 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4681 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4741 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4801 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4861 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4921 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4981 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5041 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5101 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5161 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5221 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5281 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5341 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5401 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5461 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5521 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5581 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5641 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5701 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5761 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     5821 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     5881 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     5941 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     6001 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacg
//
