LOCUS       sequence-320616-        6633 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutant of hamster BiP-mGFP unable to bind clients..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6633)
  AUTHORS   Lai CW, Aronson DE, Snapp EL
  TITLE     BiP availability distinguishes states of homeostasis and stress in 
            the endoplasmic reticulum of living cells.
  JOURNAL   Mol Biol Cell. 2010 Jun 15;21(12):1909-21. doi: 
            10.1091/mbc.E09-12-1066. Epub 2010 Apr 21.
  PUBMED    20410136
REFERENCE   2  (bases 1 to 6633)
  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: 
            "10.1091/mbc.E09-12-1066"; journalName: "Mol Biol Cell"; date: 
            "2010-06-15- 15"; volume: "21"; issue: "12"; pages: "1909-21"
COMMENT     Sequence Label: BiP-mGFP P495L
FEATURES             Location/Qualifiers
     source          1..6633
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        2..305
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        306..509
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     460..480
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2506..2515
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2512..2514,2515..2517,2518..3228)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2557..2578)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2818..2837)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3165..3186
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3362..3483
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3399..3418)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3453..3472
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3490..3945)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3627..3648)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3839..3858
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3972..4076
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4078..4435
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4078..4269
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4101..4121)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4286..4421
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4470..5264
                     /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             4470..5264
                     /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(4524..4543)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5134..5153
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5452..5471)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5496..5543
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5872..6460
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6361..6380
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
       61 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      121 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      181 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      241 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      301 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      361 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      421 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      481 tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcacc
      541 atgaagttcc ctatggtggc ggcggcgctg ctgctgctct gcgcggtgcg ggccgaggag
      601 gaggacaaga aggaggatgt gggcacggtg gtcggcatcg acctggggac cacctattcc
      661 tgcgttggtg tgttcaagaa cggccgcgtg gagatcatag ccaacgatca gggcaaccgc
      721 atcacgccgt cgtatgtggc cttcactcct gaaggcgagc gtctgattgg cgatgcggcc
      781 aagaaccagc tcacctccaa tcccgagaac acggtcttcg acgccaagcg cctcatcgga
      841 cgcacttgga atgacccttc agtgcagcag gacatcaagt tcttgccttt caaggtggtt
      901 gaaaagaaaa ctaaaccata cattcaagtt gatattggag gtgggcaaac caaaacattt
      961 gccccagaag aaatttctgc catggttctc actaaaatga aagaaactgc tgaagcatat
     1021 ttgggaaaga aggttaccca tgcagttgtt actgtgccgg cttacttcaa tgatgcccag
     1081 cgccaagcaa ccaaagatgc tggcaccatt gctggactga atgtcatgcg gatcatcaat
     1141 gagcccacag cagctgctat tgcgtatggc ctggataaga gagagggcga gaagaacatc
     1201 ctcgtttttg acctgggcgg tggaaccttc gatgtgtctc ttctgaccat tgacaatggt
     1261 gtctttgaag tggtggccac gaatggagac actcatctcg gtggggaaga ctttgatcag
     1321 cgggttatgg aacacttcat caagctgtac aaaaagaaaa ctgggaaaga cgttagaaaa
     1381 gacaacagag ctgtgcagaa acttcgtcgt gaggtggaaa aggctaagcg agccctgtct
     1441 tctcagcatc aagcaagaat tgagatagag tccttctttg aaggagaaga cttctctgag
     1501 accctgactc gggccaaatt tgaagagttg aacatggacc tgttccgatc taccatgaag
     1561 ccagtccaga aagtgttgga agactctgat ctgaagaaat cagacattga tgaaattgtt
     1621 cttgtcggtg ggtctactcg gattcccaag attcagcagc tggtgaaaga gttcttcaat
     1681 ggcaaggagc catcccgtgg cataaaccca gatgaggctg tagcatacgg tgctgctgtc
     1741 caggctggtg tcctctctgg tgatcaagat acaggtgatc tggtactgct tgatgtatgt
     1801 cctcttacac ttggtattga aacagtggga ggtgtcatga ccaaactgat tccaaggaac
     1861 actgtggtac ccaccaagaa gtctcagatc ttttccacag cttctgataa tcagccaact
     1921 gtaacaatca aggtctatga aggtgaacga cccctaacaa aagacaacca tcttctgggt
     1981 acatttgatc tgactggaat tcctcctgct cctcgtgggg tactccagat tgaagtcacc
     2041 tttgagatag atgttaatgg tattcttcga gtgacagctg aagacaaagg tacagggaac
     2101 aaaaacaaaa tcacaattac caatgaccaa aatcgcctga cacctgaaga aattgaaagg
     2161 atggttaatg atgcagagaa gtttgctgag gaagacaaaa agctcaaaga gcgcattgat
     2221 accaggaacg agttggaaag ctatgcttac tctctcaaga accagattgg agataaagaa
     2281 aagctgggcg gtaaactttc ctctgaagat aaagaaacca tggagaaagc tgtagaggaa
     2341 aagattgaat ggctggaaag ccaccaggat gcagacattg aagactttaa agctaaaaag
     2401 aaggaactag aggaaattgt tcagcctatt attagcaaac tctatggaag tgcaggccct
     2461 cccccaactg gtgaagagga tacatcagaa ggggatccac cggtcgccac catggtgagc
     2521 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     2581 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     2641 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     2701 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     2761 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     2821 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     2881 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     2941 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     3001 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     3061 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     3121 acccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     3181 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga tgaattgtaa
     3241 gcggccgcga ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt
     3301 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     3361 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     3421 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     3481 ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     3541 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     3601 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     3661 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     3721 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     3781 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     3841 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     3901 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     3961 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4021 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     4081 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     4141 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     4201 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4261 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     4321 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     4381 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     4441 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     4501 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     4561 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     4621 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     4681 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     4741 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     4801 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     4861 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     4921 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     4981 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     5041 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     5101 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     5161 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     5221 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     5281 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     5341 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     5401 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
     5461 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     5521 gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     5581 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     5641 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     5701 tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt
     5761 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5821 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5881 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5941 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6001 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6061 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6121 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6181 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6241 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6301 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6361 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6421 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6481 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     6541 tgattctgtg gataaccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg
     6601 ggtcattagt tcatagccca tatatggagt tcc
//
