LOCUS       sequence-357196-        6244 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High affinity mutant of human platelet membrane protein GP1b alpha.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6244)
  AUTHORS   Jiang Y, Fu H, Springer TA, Wong WP
  TITLE     Electrostatic Steering Enables Flow-Activated Von Willebrand Factor 
            to Bind Platelet Glycoprotein, Revealed by Single-Molecule 
            Stretching and Imaging.
  JOURNAL   J Mol Biol. 2019 Mar 29;431(7):1380-1396. doi: 
            10.1016/j.jmb.2019.02.014. Epub 2019 Feb 22.
  PUBMED    30797858
REFERENCE   2  (bases 1 to 6244)
  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: 
            "10.1016/j.jmb.2019.02.014"; journalName: "J Mol Biol"; date: 
            "2019-03-29- 29"; volume: "431"; issue: "7"; pages: "1380-1396"
COMMENT     Sequence Label: ET8-GPIba-high-6His
FEATURES             Location/Qualifiers
     source          1..6244
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1558..1575
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    join(1603..2177,2178..2180,2181..2189)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1770..1787)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1997..2016
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2190..2192,2193..2195,2196..2909)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2190..2192,2193..2195,2196..2906)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2235..2256)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2496..2515)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2843..2864
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3032..3153
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3069..3088)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3123..3142
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3160..3615)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3297..3318)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3509..3528
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3642..3746
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3748..4105
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3748..3939
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3771..3791)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3956..4091
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4140..4934
                     /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             4140..4934
                     /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(4194..4213)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4804..4823
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5122..5141)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5166..5213
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5542..6130
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6031..6050
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgtcg
      601 acgtacgcca ccatggatat gagagttcct gctcaattgc tggggttgct cttgctctgg
      661 ttcagtgggg tgttggggca ccccatctgt gaggtctcca aagtggccag ccacctagaa
      721 gtgaactgtg acaagaggaa tctgacagcg ctgcctccag acctgccgaa agacacaacc
      781 atcctccacc tgagtgagaa cctcctgtac accttctccc tggcaaccct gatgccttac
      841 actcgcctca ctcagctgaa cctagatagg tgcgagctca ccaagctcca ggtcgatggg
      901 acgctgccag tgctggggac cctggatcta tcccacaatc agctgcaaag cctgcccttg
      961 ctagggcaga cactgcctgc tctcaccgtc ctggacgtct ccttcaaccg gctgacctcg
     1021 ctgcctcttg gtgccctgcg tggtcttggt gaactccaag agctctacct gaaaggcaat
     1081 gagctgaaga ccctgccccc agggctcctg acgcccacac ccaagctgga gaagctcagt
     1141 ctggctaaca acaacttgac tgagctcccc gctgggctcc tgaatgggct ggagaatctc
     1201 gacacccttc tcctccaaga gaactcgctg tatacaatac caaagggctt ttttgggtcc
     1261 cacctcctgc cttttgcttt tctccacggg aacccctggt tatgcaactg tgagatcctc
     1321 tattttcgtc gctggctgca ggacaatgct gaaaatgtct acgtatggaa gcaagtggtg
     1381 gacgtcaagg ccgtgacctc taacgtggcc agtgtgcagt gtgacaattc agacaagttt
     1441 cccgtctaca aatacccagg aaaggggtgc cccacccttg gtgatgaagg tgacacagac
     1501 ctatatgatt actacccaga agaggacact gagggcgata aggtgcgtgg cagcggccat
     1561 caccatcacc atcacgcgta aactagtggc gcgccggatc cgcccctctc cctccccccc
     1621 ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt
     1681 attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
     1741 cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
     1801 tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac
     1861 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg
     1921 tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt
     1981 tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca
     2041 gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tacacatgct ttacatgtgt
     2101 ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg
     2161 aaaaacacga tgataatatg gccacaacca tggtgagcaa gggcgaggag ctgttcaccg
     2221 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     2281 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     2341 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     2401 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     2461 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     2521 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     2581 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     2641 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     2701 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2761 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     2821 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     2881 ctctcggcat ggacgagctg tacaagtaaa gcggccgcga ctctagatca taatcagcca
     2941 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     3001 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     3061 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3121 ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt
     3181 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     3241 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     3301 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     3361 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     3421 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     3481 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     3541 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     3601 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3661 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3721 ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg
     3781 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3841 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3901 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3961 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     4021 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     4081 ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca
     4141 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     4201 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     4261 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     4321 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     4381 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     4441 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     4501 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4561 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4621 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     4681 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4741 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     4801 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4861 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4921 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     4981 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     5041 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     5101 ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
     5161 tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg
     5221 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     5281 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     5341 gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact
     5401 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     5461 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5521 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5581 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5641 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     5701 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5761 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5821 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5881 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5941 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6001 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6061 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6121 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6181 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat
     6241 gcat
//
