LOCUS       sequence-424182-        6390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of a gene, accompanied by the expression of the 
            affinity sorting tag GST-EGFP-GPICEAM7 driven by an SV40 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6390)
  AUTHORS   Ma S, Yang L, Zuo Q, Huang Q
  TITLE     GPI-anchored glutathione S-transferase as marker allows affinity 
            sorting of transfection-positive cells.
  JOURNAL   Front Mol Biosci. 2022 Sep 29;9:1016090. doi: 
            10.3389/fmolb.2022.1016090. eCollection 2022.
  PUBMED    36250010
REFERENCE   2  (bases 1 to 6390)
  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: "Front Mol 
            Biosci."; date: "2022-09-29"; volume: "9:1016090. doi"; pages: " 
            10.3389/fmolb.2022.1016090. eCollection 2022"
COMMENT     Sequence Label: pcDNA3.1-GST-EGFP-GPICEAM7
FEATURES             Location/Qualifiers
     source          1..6390
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1000..1041
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1051..1068
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1091..1108)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1097..1321
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1367..1795
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1454..1473)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1664..1685
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1804..1824)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1809..2138
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1989..2124
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2051..2070
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2334..2990
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MGSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2948..2970
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2997..3014
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      3021..3030
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3027..3029,3030..3032,3033..3743)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3027..3029,3030..3032,3033..3743)
                     /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(3072..3093)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3333..3352)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3680..3701
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4063..4184
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4100..4119)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4154..4173
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4233..4249)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4233..4249)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4246..4268)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4257..4273
                     /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(4281..4287,4288..4305,4306..4311))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4326..4347
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4464..4481)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4635..5223)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4715..4734)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5394..6185,6186..6254))
                     /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     6017..6036
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6255..6359)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagcttggt accgagctcg gatccactag tccagtgtgg tggaattctg cagatatcca
      961 gcacagtggc ggccgctcga gtctagaggg cccttcgaag gtaagcctat ccctaaccct
     1021 ctcctcggtc tcgattctac gcgtaccggt catcatcacc atcaccattg agtttaaacc
     1081 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     1141 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     1201 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     1261 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     1321 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     1381 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1441 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     1501 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     1561 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     1621 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     1681 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     1741 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     1801 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     1861 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     1921 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     1981 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     2041 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     2101 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     2161 ccattttcgg atctgatcaa gagacaggat gaggatcgtt taccatgggc agccctagcg
     2221 cctgccctta cagagtgtgc atcccctggc agggcctgct gctgaccgcc agcctgctga
     2281 ccttctggaa cctgcccaac agcgcccaga ccagcgcctg gagccaccct cagatgggct
     2341 cccctatact aggttattgg aaaattaagg gccttgtgca acccactcga cttcttttgg
     2401 aatatcttga agaaaaatat gaagagcatt tgtatgagcg cgatgaaggt gataaatggc
     2461 gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct tccttattat attgatggtg
     2521 atgttaaatt aacacagtct atggccatca tacgttatat agctgacaag cacaacatgt
     2581 tgggtggttg tccaaaagag cgtgcagaga tttcaatgct tgaaggagcg gttttggata
     2641 ttagatacgg tgtttcgaga attgcatata gtaaagactt tgaaactctc aaagttgatt
     2701 ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt cataaaacat
     2761 atttaaatgg tgatcatgta acccatcctg acttcatgtt gtatgacgct cttgatgttg
     2821 ttttatacat ggacccaatg tgcctggatg cgttcccaaa attagtttgt tttaaaaaac
     2881 gtattgaagc tatcccacaa attgataagt acttgaaatc cagcaagtat atagcatggc
     2941 ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca tcctccaaaa tcggatctgg
     3001 ttccgcgtgg atccccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     3061 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     3121 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     3181 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     3241 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     3301 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     3361 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     3421 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     3481 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     3541 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3601 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     3661 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3721 tcggcatgga cgagctgtac aagtccggcc ggactcagat cagccggagc gaccctgtga
     3781 ccctgaacgt gagatacgag agcgtgcagg ccagcagccc tgacctgagc gccggcaccg
     3841 ccgtgagcat catgatcggc gtgctggctg gcatggctct gatctaggcg ggactctggg
     3901 gttcgcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     3961 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4021 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     4081 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4141 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     4201 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4261 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4321 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4381 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4441 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4501 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4561 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4621 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4681 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4741 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4801 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4861 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4921 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4981 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5041 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5101 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5161 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5221 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5281 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5341 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5401 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5461 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5521 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5581 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5641 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5701 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5761 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5821 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5881 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5941 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6001 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6061 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6121 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6181 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6241 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6301 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6361 acatttcccc gaaaagtgcc acctgacgtc
//
