LOCUS       sequence-177624-        4728 bp    DNA     circular SYN 14-APR-2025
DEFINITION  To express EGFP-6xHis in mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4728)
  AUTHORS   Tie HC, Madugula V, Lu L
  TITLE     The development of a single molecule fluorescence standard and its 
            application in estimating the stoichiometry of the nuclear pore 
            complex.
  JOURNAL   Biochem Biophys Res Commun. 2016 Sep 30;478(4):1694-9. doi: 
            10.1016/j.bbrc.2016.09.005. Epub 2016 Sep 6.
  PUBMED    27613095
REFERENCE   2  (bases 1 to 4728)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.bbrc.2016.09.005"; journalName: "Biochem Biophys Res 
            Commun"; date: "2016-09-30- 30"; volume: "478"; issue: "4"; pages: 
            "1694-9"
COMMENT     Sequence Label: pEGFP-C1-6xHis
FEATURES             Location/Qualifiers
     source          1..4728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      134..722
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     623..642
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        897..1200
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1201..1404
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1355..1375
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1443..1452
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1449..1451,1452..1454,1455..2165)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1449..1451,1452..1454,1455..2165)
                     /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(1494..1515)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1755..1774)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2102..2123
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2202..2219
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    2352..2473
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2389..2408)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2443..2462
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2480..2935)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2617..2638)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2829..2848
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2962..3066
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3068..3425
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3068..3259
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3091..3111)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3276..3411
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3460..4254
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3460..4254
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3514..3533)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4124..4143
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4442..4461)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4486..4533
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
       61 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
      121 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
      181 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
      241 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
      301 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
      361 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
      421 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
      481 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
      541 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
      601 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
      661 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
      721 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
      781 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcattagt
      841 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      901 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      961 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     1021 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     1081 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     1141 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     1201 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     1261 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     1321 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     1381 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
     1441 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     1501 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     1561 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     1621 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     1681 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     1741 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     1801 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     1861 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     1921 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     1981 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2041 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     2101 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     2161 acaagtccgg actcagatct cgagctcaag cttcgaattc tcaccaccac caccaccact
     2221 gaggatccac cggatctaga taactgatca taatcagcca taccacattt gtagaggttt
     2281 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     2341 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     2401 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     2461 tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     2521 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     2581 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     2641 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     2701 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     2761 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     2821 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     2881 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     2941 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3001 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3061 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3121 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3181 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3241 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3301 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3361 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3421 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3481 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3541 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3601 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     3661 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3721 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3781 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3841 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     3901 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     3961 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     4021 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4081 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4141 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4201 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4261 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4321 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4381 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa
     4441 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     4501 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct
     4561 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct
     4621 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg
     4681 cggcaggccc tgccatagcc tcaggttact catatatact ttagattg
//
