LOCUS       sequence-306308-        8573 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of 3a-EGFP with CMV promoter, can be used for 
            transfection or used to generate bacmids for baculoviral infection 
            of mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8573)
  AUTHORS   Kern DM, Sorum B, Mali SS, Hoel CM, Sridharan S, Remis JP, Toso DB, 
            Kotecha A, Bautista DM, Brohawn SG
  TITLE     Cryo-EM structure of the SARS-CoV-2 3a ion channel in lipid 
            nanodiscs.
  JOURNAL   bioRxiv [Preprint]. 2021 Jan 26:2020.06.17.156554. doi: 
            10.1101/2020.06.17.156554.
  PUBMED    32587976
REFERENCE   2  (bases 1 to 8573)
  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: "bioRxiv 
            [Preprint]. 2021 Jan 26:2020.06.17.156554. doi: 
            10.1101/2020.06.17.156554."
COMMENT     Sequence Label: pSBL_DK186
FEATURES             Location/Qualifiers
     source          1..8573
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      102..556
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(189..208)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     398..419
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        582..686
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(687..755,756..1547)
                     /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     complement(905..924)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1718..2306
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2207..2226
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2460..2477
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2611..2835
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2902..3435)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3624..3629,3630..3646,3647..3652))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    4169..4217
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4242..4261
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(4357..4466)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        4511..4602
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     4527..4544
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     enhancer        4782..5161
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5162..5365
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5316..5336
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5362..5386
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          5501..5633
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     5616..5635
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      5697..5706
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6537..6560
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(6576..6578,6579..7289)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6618..6639)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6879..6898)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7226..7247
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             7302..7331
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             7342..7371
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_feature    7442..8030
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7495..7515)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7913..7924)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    8163..8297
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8200..8219)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8254..8273
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  8326..8491
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga
       61 ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
      121 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
      181 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
      241 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
      301 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
      361 ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt ccaaactgga
      421 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
      481 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
      541 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
      601 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
      661 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
      721 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
      781 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
      841 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
      901 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
      961 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     1021 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     1081 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     1141 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     1201 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     1261 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     1321 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     1381 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     1441 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     1501 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     1561 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     1621 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     1681 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     1741 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     1801 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     1861 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     1921 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     1981 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2041 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2101 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2161 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2221 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2281 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2341 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2401 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2461 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     2521 tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg gttacggttg
     2581 agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa
     2641 caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga
     2701 aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact
     2761 cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg
     2821 gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc
     2881 gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac
     2941 ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc
     3001 ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag
     3061 cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat
     3121 ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc caacaaccgc
     3181 ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta
     3241 atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag
     3301 atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat
     3361 gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt
     3421 gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg
     3481 cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga
     3541 aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg
     3601 atcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc
     3661 gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg
     3721 aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg
     3781 cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg cccttgcttc
     3841 aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag
     3901 tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct
     3961 atagttctag tggttggcta cagctttgtt taaacaaagc tggctatggc agggcttgcc
     4021 gccccgacgt tggctgcgag ccctgggcct tcacccgaac ttgggggttg gggtggggaa
     4081 aaggaagaaa cgcgggcgta ttggtcccaa tggggtctcg gtggggtatc gacagagtgc
     4141 cagccctggg accgaacccc gcgtttatga acaaacgacc caacacccgt gcgttttatt
     4201 ctgtcttttt attgccgtca tagcgcgggt tccttccggt attgtctcct tccgtgtttc
     4261 agttagcctc ccccatctcc cggtaccgca tgctatgcat cagctgctag caccatggct
     4321 cgatcgagat cccgggtgat caagtcttcg tcgagtgatt gtaaataaaa tgtaatttac
     4381 agtatagtat tttaattaat atacaaatga tttgataata attcttattt aactataata
     4441 tattgtgttg ggttgaatta aaggtccgta tactagtatc gattcgcgac ctactccgga
     4501 atattaatag atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt
     4561 tactgttttc gtaacagttt tgtaataaaa aaacctataa atattccgga ttattcatac
     4621 cgtcccacca tcgggcgcgg atcctcaata ttggccatta gccatattat tcattggtta
     4681 tatagcataa atcaatattg gctattggcc attgcatacg ttgtatctat atcataatat
     4741 gtacatttat attggctcat gtccaatatg accgccatgt tggcattgat tattgactag
     4801 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     4861 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     4921 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     4981 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     5041 tccgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     5101 gaccttacgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     5161 ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag cggtttgact cacggggatt
     5221 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     5281 ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa atgggcggta ggcgtgtacg
     5341 gtgggaggtc tatataagca gagctcgttt agtgaaccgt cagatcacta gaagctttat
     5401 tgcggtagtt tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctc
     5461 gaacttaagc tgcagaagtt ggtcgtgagg cactgggcag gtaagtatca aggttacaag
     5521 acaggtttaa ggagaccaat agaaactggg cttgtcgaga cagagaagac tcttgcgttt
     5581 ctgataggca cctattggtc ttactgacat ccactttgcc tttctctcca caggtgtcca
     5641 ctcccagttc aattacagct cttaagggat cccggtccga agcgcgctag cctcgagcca
     5701 ccatggatct ctttatgcgc atttttacga tcggtacagt tacgttgaag caaggtgaaa
     5761 tcaaagacgc cacgccgtcc gattttgttc gcgctacggc tacaattcca atccaagcga
     5821 gcttgccatt cggttggttg atagttggtg ttgcgttgct tgcagtcttt cagtcagctt
     5881 ctaagataat cactctgaag aagcgttggc aactggcgct ctcaaaaggc gttcacttcg
     5941 tgtgcaacct cctgttgctc tttgtcaccg tttactcaca tctcttgctt gttgctgctg
     6001 gcctggaagc tcccttcctt tacctgtacg ctctggtcta ttttctgcag agcataaatt
     6061 ttgtcaggat aataatgaga ctgtggcttt gttggaaatg ccgctccaag aacccgcttt
     6121 tgtacgatgc caattacttc ctgtgttggc atacgaattg ctacgactac tgtattccgt
     6181 ataatagcgt cacttcttca atcgtgataa cgtccggtga cggtacaaca tcacctattt
     6241 ctgaacacga ttaccaaata ggtggctaca ccgagaaatg ggagagtgga gtcaaagatt
     6301 gtgtagtgct gcactcatat tttacctcgg attattatca actctactca acacagttgt
     6361 ctactgatac aggagttgag cacgtaacat ttttcattta caataaaatt gtggacgaac
     6421 ccgaggagca tgtacagatt catactatcg acggtagcag cggtgtcgtg aaccccgtta
     6481 tggagccaat ttacgacgaa cccaccacga caacatccgt ccccctgtcg aattctttgg
     6541 aagttttgtt tcaaggtcca actgctgccg ccgctgtgag caagggcgag gagctgttca
     6601 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     6661 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     6721 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     6781 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     6841 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     6901 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     6961 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     7021 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     7081 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     7141 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca
     7201 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     7261 tcactctcgg catggacgag ctgtacaagt ccggaggtgg tcaccatcac catcaccacc
     7321 atcaccatca ctaatagtct agaacaaaaa ctcatctcag aagaggatct gaatagcgcc
     7381 gtcgacgagc tcactagtcg cggccgcttt cgaatctaga gcctgcagtc tcgacaagct
     7441 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     7501 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     7561 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     7621 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     7681 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     7741 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     7801 gttgggcact gacaattccg tggtgttgtc ggggaagctg acgtcctttc catggctgct
     7861 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     7921 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     7981 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc ctgaagcttg
     8041 tcgagaagta ctagaggatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     8101 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     8161 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     8221 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     8281 cttatcatgt ctggatctga tcactgcttg agcctaggag atccgaacca gataagtgaa
     8341 atctagttcc aaactatttt gtcattttta attttcgtat tagcttacga cgctacaccc
     8401 agttcccatc tattttgtca ctcttcccta aataatcctt aaaaactcca tttccacccc
     8461 tcccagttcc caactatttt gtccgcccac agcggggcat ttttcttcct gttatgtttt
     8521 taatcaaaca tcctgccaac tccatgtgac aaaccgtcat cttcggctac ttt
//
