LOCUS       sequence-384487-        5813 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for GFP tagged NHERF1. Triple PKC site 
            phosphomimetic mutant (S162/339/340D).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5813)
  AUTHORS   Garbett D, LaLonde DP, Bretscher A
  TITLE     The scaffolding protein EBP50 regulates microvillar assembly in a 
            phosphorylation-dependent manner.
  JOURNAL   J Cell Biol. 2010 Oct 18;191(2):397-413. doi: 10.1083/jcb.201004115.
            Epub 2010 Oct 11.
  PUBMED    20937695
REFERENCE   2  (bases 1 to 5813)
  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: 
            "10.1083/jcb.201004115"; journalName: "J Cell Biol"; date: 
            "2010-10-18- 18"; volume: "191"; issue: "2"; pages: "397-413"
COMMENT     Sequence Label: GFP-NHERF1 3xSD
FEATURES             Location/Qualifiers
     source          1..5813
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        74..377
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        378..581
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     532..552
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      620..629
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(626..628,629..631,632..1342)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(626..628,629..631,632..1342)
                     /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(671..692)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(932..951)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1279..1300
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2614..2735
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2651..2670)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2705..2724
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2742..3197)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2879..2900)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3091..3110
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3224..3328
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3330..3687
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3330..3521
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3353..3373)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3538..3673
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3722..4516
                     /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             3722..4516
                     /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(3776..3795)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4386..4405
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4704..4723)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4748..4795
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5124..5712
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5613..5632
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taccgccatg cattagttat taatagtaat caattacggg gtcattagtt catagcccat
       61 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      121 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      181 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      241 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      301 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      361 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      421 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      481 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      541 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga
      601 tccgctagcg ctaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt
      661 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
      721 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
      781 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
      841 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
      901 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
      961 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     1021 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     1081 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     1141 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     1201 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     1261 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     1321 cggcatggac gagctgtaca agtccggccg gactcagatc ctgatgagcg cggacgcagc
     1381 ggccggggcg cccctgcccc ggctctgctg cctggagaag ggtccgaacg gctacggctt
     1441 ccacctgcac ggggagaagg gcaagttggg ccagtacatc cggctggtgg agcccggctc
     1501 gccggccgag aaggcggggc tgctggcggg ggaccggctg gtggaggtga acggggagaa
     1561 cgtggaaaag gagacccacc agcaggtggt gagccgcatc cgcgccgcac tcaacgccgt
     1621 gcgcctgctg gtggtcgacc ccgagacgga cgagcagctg cagaagctcg gcgtccaggt
     1681 ccgagaggag ctgctgcgcg cccaggaagc gccggggcag gccgagccgc cggccgccgc
     1741 cgaggtgcag ggggctggca acgaaaatga gcctcgcgag gccgacaaga gccacccgga
     1801 gcagcgcgag cttcggcctc ggctctgtac catgaagaag ggccccgacg gctatggctt
     1861 caacctgcac agcgacaagt ccaagccagg ccagttcatc cggtcagtgg acccagactc
     1921 cccggctgag gcttcagggc tccgggccca ggatcgcatt gtggaggtga acggggtctg
     1981 catggagggg aagcagcatg gggacgtggt gtccgccatc agggctggcg gggacgagac
     2041 caagctgctg gtggtggaca gggaaactga cgagttcttc aagaaatgca gagtgatccc
     2101 atctcaggag cacctgaatg gtcccctgcc tgtgcccttc accaatgggg agatacagaa
     2161 ggagaacagt cgtgaagccc tggcagaggc agccttggag agccccaggc cagccctggt
     2221 gagatccgcc tccagtgaca ccagcgagga gctgaattcc caagacagcc ccccaaaaca
     2281 ggactccaca gcgccctcgt ctacctcctc ctccgacccc atcctagact tcaacatctc
     2341 cctggccatg gccaaagaga gggcccacca gaaacgcgac gacaaacggg ccccgcagat
     2401 ggactggagc aagaaaaacg aactcttcag caacctctga gccgggtcga ggggaattct
     2461 gcagtcgacg gtaccgcggg cccgggatcc accggatcta gataactgat cataatcagc
     2521 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     2581 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     2641 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     2701 agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat
     2761 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     2821 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     2881 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     2941 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     3001 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     3061 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     3121 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     3181 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3241 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3301 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg
     3361 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3421 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3481 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3541 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3601 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     3661 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg
     3721 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3781 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3841 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3901 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3961 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4021 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4081 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4141 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4201 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     4261 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4321 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4381 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4441 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4501 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     4561 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4621 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4681 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     4741 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
     4801 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     4861 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     4921 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
     4981 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5041 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5101 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5161 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5221 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     5281 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5341 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5401 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5461 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5521 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5581 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5641 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5701 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5761 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tat
//
