LOCUS       sequence-422961-        5793 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of a gene, accompanied by the expression of the 
            affinity sorting tag TST-EGFP-GPIDAF driven by an SV40 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5793)
  AUTHORS   Yang L, Cui L, Ma S, Zuo Q, Huang Q
  TITLE     A Gene Transfer-Positive Cell Sorting System Utilizing 
            Membrane-Anchoring Affinity Tag.
  JOURNAL   Front Bioeng Biotechnol. 2022 Jun 16;10:930966. doi: 
            10.3389/fbioe.2022.930966. eCollection 2022.
  PUBMED    35782508
REFERENCE   2  (bases 1 to 5793)
  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 
            Bioeng Biotechnol."; date: "2022-06-16"; volume: "10:930966. doi"; 
            pages: " 10.3389/fbioe.2022.930966. eCollection 2022"
COMMENT     Sequence Label: pcDNA3.1-TST-EGFP-GPIDAF
FEATURES             Location/Qualifiers
     source          1..5793
                     /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             join(2307..2312,2313..2336,2337..2372,2373..2396)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     regulatory      2403..2412
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2409..2411,2412..2414,2415..3125)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2409..2411,2412..2414,2415..3125)
                     /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(2454..2475)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2715..2734)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3062..3083
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3466..3587
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3503..3522)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3557..3576
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3636..3652)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3636..3652)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3649..3671)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3660..3676
                     /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(3684..3690,3691..3708,3709..3714))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3729..3750
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3867..3884)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4038..4626)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4118..4137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4797..5588,5589..5657))
                     /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     5420..5439
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5658..5762)
                     /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 taccatgacc gtggctagac
     2221 ccagcgtgcc tgctgccctg cctctgctgg gagagctgcc tagactgctg ctcctggtgc
     2281 tcctgtgcct gcctgctgtg tggggaagcg cctggagcca ccctcagttc gagaaaggag
     2341 gaggcagcgg cggcggaagc ggaggctccg cttggtccca cccacagttc gaaaagccgg
     2401 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     2461 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     2521 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     2581 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     2641 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     2701 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     2761 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     2821 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     2881 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     2941 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     3001 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     3061 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     3121 acaagtccgg ccggactcag atcagccgga ccaccaagca cttccacgag accaccccta
     3181 acaagggcag cggcaccacc agcggcacca cccggctgct gagcggccac acctgcttca
     3241 ccctgaccgg cctgctgggc accctggtga ccatgggcct gctgacctga gcgggactct
     3301 ggggttcgcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     3361 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     3421 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     3481 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3541 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     3601 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3661 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3721 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3781 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3841 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3901 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3961 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4021 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4081 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4141 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4201 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4261 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4321 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4381 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4441 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4501 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4561 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4621 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4681 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4741 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4801 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4861 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4921 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4981 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5041 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5101 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5161 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5221 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5281 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5341 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5401 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5461 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5521 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5581 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5641 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5701 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5761 cgcacatttc cccgaaaagt gccacctgac gtc
//
