LOCUS       sequence-319257-        7690 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector optimized for use in screening assays, as well as for 
            efficient production of baculovirus and robust expression of the 
            target protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7690)
  AUTHORS   Goehring A, Lee CH, Wang KH, Michel JC, Claxton DP, Baconguis I, 
            Althoff T, Fischer S, Garcia KC, Gouaux E
  TITLE     Screening and large-scale expression of membrane proteins in 
            mammalian cells for structural studies.
  JOURNAL   Nat Protoc. 2014 Nov;9(11):2574-85. doi: 10.1038/nprot.2014.173. 
            Epub 2014 Oct 9.
  PUBMED    25299155
REFERENCE   2  (bases 1 to 7690)
  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: 
            "10.1038/nprot.2014.173"; journalName: "Nat Protoc"; date: 
            "2014-11"; volume: "9"; issue: "11"; pages: "2574-85"
COMMENT     Sequence Label: pEG BacMam C term 3C eGFP His8
FEATURES             Location/Qualifiers
     source          1..7690
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     342..361
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     595..612
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  746..970
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(1037..1570)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(1759..1764,1765..1781,1782..1787))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    2304..2352
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     2377..2396
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(2488..2597)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        2642..2733
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     2658..2675
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     enhancer        2913..3292
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3293..3496
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3447..3467
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3493..3517
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          3632..3764
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3747..3766
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             3876..3899
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(3912..3914,3915..4625)
                     /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(3954..3975)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4215..4234)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4562..4583
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4641..4664
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     misc_feature    4694..5282
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4747..4767)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5165..5176)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5415..5549
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5452..5471)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5506..5525
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  5578..5743
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      5927..6381
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6014..6033)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6223..6244
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6407..6511
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6512..6580,6581..7372)
                     /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(6730..6749)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7543..7690,1..441)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
       61 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
      121 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
      181 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
      241 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
      301 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
      361 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
      421 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
      481 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
      541 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
      601 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
      661 ggtatttcac accgcataga ccagccgcgt aacctggcaa aatcggttac ggttgagtaa
      721 taaatggatg ccctgcgtaa gcgggtgtgg gcggacaata aagtcttaaa ctgaacaaaa
      781 tagatctaaa ctatgacaat aaagtcttaa actagacaga atagttgtaa actgaaatca
      841 gtccagttat gctgtgaaaa agcatactgg acttttgtta tggctaaagc aaactcttca
      901 ttttctgaag tgcaaattgc ccgtcgtatt aaagaggggc gtggccaagg gcatggtaaa
      961 gactatattc gcggcgttgt gacaatttac cgaacaactc cgcggccggg aagccgatct
     1021 cggcttgaac gaattgttag gtggcggtac ttgggtcgat atcaaagtgc atcacttctt
     1081 cccgtatgcc caactttgta tagagagcca ctgcgggatc gtcaccgtaa tctgcttgca
     1141 cgtagatcac ataagcacca agcgcgttgg cctcatgctt gaggagattg atgagcgcgg
     1201 tggcaatgcc ctgcctccgg tgctcgccgg agactgcgag atcatagata tagatctcac
     1261 tacgcggctg ctcaaacttg ggcagaacgt aagccgcgag agcgccaaca accgcttctt
     1321 ggtcgaaggc agcaagcgcg atgaatgtct tactacggag caagttcccg aggtaatcgg
     1381 agtccggctg atgttgggag taggtggcta cgtctccgaa ctcacgaccg aaaagatcaa
     1441 gagcagcccg catggatttg acttggtcag ggccgagcct acatgtgcga atgatgccca
     1501 tacttgagcc acctaacttt gttttagggc gactgccctg ctgcgtaaca tcgttgctgc
     1561 tgcgtaacat cgttgctgct ccataacatc aaacatcgac ccacggcgta acgcgcttgc
     1621 tgcttggatg cccgaggcat agactgtaca aaaaaacagt cataacaagc catgaaaacc
     1681 gccactgcgc cgttaccacc gctgcgttcg gtcaaggttc tggaccagtt gcgtgatcgc
     1741 atacgctact tgcattacag tttacgaacc gaacaggctt atgtcaactg ggttcgtgcc
     1801 ttcatccgtt tccacggtgt gcgtcacccg gcaaccttgg gcagcagcga agtcgaggca
     1861 tttctgtcct ggctggcgaa cgagcgcaag gtttcggtct ccacgcatcg tcaggcattg
     1921 gcggccttgc tgttcttcta cggcaaggtg ctgtgcacgg atctgccctt gcttcaggag
     1981 atcggtagac ctcggccgtc gcggcgcttg ccggtggtgc tgaccccgga tgaagtggtt
     2041 cgcatcctcg gttttctgga aggcgagcat cgtttgttcg cccaggactc tagctatagt
     2101 tctagtggtt ggctacagct ttgtttaaac aaagctggct atggcagggc ttgccgcccc
     2161 gacgttggct gcgagccctg ggccttcacc cgaacttggg ggttggggtg gggaaaagga
     2221 agaaacgcgg gcgtattggt cccaatgggg tctcggtggg gtatcgacag agtgccagcc
     2281 ctgggaccga accccgcgtt tatgaacaaa cgacccaaca cccgtgcgtt ttattctgtc
     2341 tttttattgc cgtcatagcg cgggttcctt ccggtattgt ctccttccgt gtttcagtta
     2401 gcctccccca tctcccggta ccgcatgcta tgcatcagct gctagcacca tggctcgaga
     2461 tcccgggtga tcaagtcttc gtcgagtgat tgtaaataaa atgtaattta cagtatagta
     2521 ttttaattaa tatacaaatg atttgataat aattcttatt taactataat atattgtgtt
     2581 gggttgaatt aaaggtccgt atactagtat cgattcgcga cctactccgg aatattaata
     2641 gatcatggag ataattaaaa tgataaccat ctcgcaaata aataagtatt ttactgtttt
     2701 cgtaacagtt ttgtaataaa aaaacctata aatattccgg attattcata ccgtcccacc
     2761 atcgggcgcg gatcctcaat attggccatt agccatatta ttcattggtt atatagcata
     2821 aatcaatatt ggctattggc cattgcatac gttgtatcta tatcataata tgtacattta
     2881 tattggctca tgtccaatat gaccgccatg ttggcattga ttattgacta gttattaata
     2941 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     3001 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     3061 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     3121 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtccgccccc
     3181 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttacg
     3241 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     3301 gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     3361 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     3421 atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt
     3481 ctatataagc agagctcgtt tagtgaaccg tcagatcact agaagcttta ttgcggtagt
     3541 ttatcacagt taaattgcta acgcagtcag tgcttctgac acaacagtct cgaacttaag
     3601 ctgcagaagt tggtcgtgag gcactgggca ggtaagtatc aaggttacaa gacaggttta
     3661 aggagaccaa tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt tctgataggc
     3721 acctattggt cttactgaca tccactttgc ctttctctcc acaggtgtcc actcccagtt
     3781 caattacagc tcttaaggga tcccggtccg aagcgcgcgg aattcaaagg cctacgtcga
     3841 cgagctcact agtcgcggcc gctttcgaat ctagactgga agttctgttc cagggtcctg
     3901 cagccgccgc ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     3961 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     4021 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     4081 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     4141 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     4201 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     4261 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     4321 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     4381 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     4441 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     4501 acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc
     4561 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     4621 acaagtccgg aggttcagga caccatcatc atcaccacca tcactgacta gagcctgcag
     4681 tctcgacaag cttaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     4741 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     4801 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     4861 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     4921 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     4981 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     5041 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt
     5101 tccatggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     5161 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     5221 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     5281 gcctgaagct tgtcgagaag tactagagga tcataatcag ccataccaca tttgtagagg
     5341 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     5401 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5461 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5521 tcatcaatgt atcttatcat gtctggatct gatcactgct tgagcctagg agatccgaac
     5581 cagataagtg aaatctagtt ccaaactatt ttgtcatttt taattttcgt attagcttac
     5641 gacgctacac ccagttccca tctattttgt cactcttccc taaataatcc ttaaaaactc
     5701 catttccacc cctcccagtt cccaactatt ttgtccgccc acagcggggc atttttcttc
     5761 ctgttatgtt tttaatcaaa catcctgcca actccatgtg acaaaccgtc atcttcggct
     5821 actttttctc tgtcacagaa tgaaaatttt tctgtcatct cttcgttatt aatgtttgta
     5881 attgactgaa tatcaacgct tatttgcagc ctgaatggcg aatgggacgc gccctgtagc
     5941 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     6001 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     6061 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     6121 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     6181 acggtttttc gccctttgac gttggagtcc acgttcttaa tagtggactc ttgttccaaa
     6241 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     6301 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     6361 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     6421 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     6481 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     6541 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     6601 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     6661 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     6721 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     6781 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     6841 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     6901 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     6961 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     7021 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     7081 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     7141 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     7201 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     7261 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     7321 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     7381 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     7441 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacatg agttttcgtt
     7501 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     7561 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     7621 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     7681 aaatactgtt
//
