LOCUS       sequence-487636-        7332 bp    DNA     circular SYN 01-OCT-2025
DEFINITION  Expression and purification of human wild-type PLEKHM1 in cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7332)
  TITLE     Martin lab plasmids
REFERENCE   2  (bases 1 to 7332)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 1, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: twin-step HRV3C PLEKHM1
FEATURES             Location/Qualifiers
     source          1..7332
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      547..556
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(559..564,565..588,589..624,625..648)
                     /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"
     CDS             667..690
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             691..714
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    4060..4181
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4097..4116)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4151..4170
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4188..4643)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4325..4346)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4537..4556
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4670..4774
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4776..5133
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4776..4967
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4799..4819)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4984..5119
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5168..5962
                     /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             5168..5962
                     /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(5222..5241)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5832..5851
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6150..6169)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6194..6241
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6570..7158
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7059..7078
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      541 ccggtcgcca ccatgggcag cgcatggagt catcctcaat tcgagaaagg tggaggttct
      601 ggcggtggat cgggaggttc agcgtggagc cacccgcagt tcgaaaaatc cggaggtgct
      661 tctggtctgg aagttctgtt ccagggaccc gattacaagg atgacgacga taagtccgga
      721 ctcagatctc gagctcaagc ttcgatgctt tcagtggtgg agaatggact ggacccccag
      781 gctgccatcc cggtcatcaa gaagaagctg gtgggatccg tgaaggcctt gcagaagcag
      841 tacgtgtccc tggacacggt ggtcactagt gaagacggag atgccaacac catgtgcagc
      901 gccctggagg ccgtatttat ccatggcctg cacgccaagc acatccgagc tgaggccgga
      961 ggaaaaagga agaaaagtgc ccaccagaag cctctgcccc agcctgtctt ctggcccctc
     1021 ctgaaagctg tcacccacaa acacatcatc tcagagttgg agcacctgac gtttgtcaac
     1081 acggatgtgg gccgctgccg ggcatggctg cggctggccc tgaacgatgg cctgatggag
     1141 tgctacctga agctgctgct gcaggagcag gcccgcttgc atgagtacta ccagcccacc
     1201 gccctgctcc gggatgctga ggagggcgag ttcctcctta gcttcctgca gggcctcacg
     1261 tccttgtcct tcgaactctc ctacaagtct gccatcttaa atgagtggac gctcacccca
     1321 ttggccctgt ctgggctttg cccgctttct gagctggacc ctctctctac ctctggtgca
     1381 gaactacagc ggaaggaatc tctggattcc atttcccatt cttcaggctc tgaagacatc
     1441 gaagtccatc actcgggcca taagatacgg aggaaccaga agctgactgc ctcctccctc
     1501 agcctggaca cggccagttc atcccagctg tcctgcagcc taaactctga tagctgctta
     1561 ctccaagaga atggctccaa gagtccagac cattgcgagg agcccatgtc ctgtgactca
     1621 gacctgggca cagcaaatgc tgaggactca gaccggtctc tgcaagaggt attgttggaa
     1681 ttcagcaaag cccaggtaaa ctctgtgcca accaacggac tgagccaaga aacagagatc
     1741 cccacaccac aggcctcgct ctccctccat ggcctcaaca ccagcacata cctgcactgt
     1801 gaggcacctg cagagcccct tcctgcccag gcagcctctg gaactcaaga tggtgtccac
     1861 gtgcaggagc cgcgtcccca ggcgcccagc cccctggact tacagcagcc tgtagagagc
     1921 acctcaggcc agcagccttc tagtactgtc agcgagacag ccagagaagt gggccaaggg
     1981 aatggcctgc agaaggccca ggctcatgac ggagctggtc tgaagctggt agtttcctca
     2041 cccaccagtc cgaaaaacaa gagctggatc tcagaggatg acttctaccg gccttcccgg
     2101 gagcaacccc tggagagtgc ttcagaccac ccaatagctt cttacagggg gactccaggg
     2161 tcaaggcctg gtctccacag gcatttttct caagaaccaa gaaaaaactg ctccctgggg
     2221 gcgttagacc aagcgtgtgt accttcccca ggaagaaggc aagcccaggc agccccatcc
     2281 caggggcata agagcttccg ggtggtacac cggagacaga tgggactgtc caacccattc
     2341 cggggtctca tgaagctggg caccgtggag cggcgggggg caatgggcat ctggaaggag
     2401 ctcttctgcg agctctcccc gctggagttc cgcctctacc tgagcaacga ggagcacacc
     2461 tgtgtggaga actgctcgct gcttcgctgt gagtctgtgg ggccagccca tagtgatggg
     2521 cgctttgagc tggtcttctc tggcaagaag ctggccctgc gcgcctcctc ccaggacgaa
     2581 gctgaggact ggctggaccg ggtgcgggag gccctgcaga aggtccggcc tcagcaggag
     2641 gatgagtggg tgaacgtgca gtacccagac cagcctgagg aaccccccga ggcgccccag
     2701 ggctgcctct ctccctcaga cctgctctcg gagcccgcgg ccctccaggg cacacagttt
     2761 gactggtcgt ccgcccaggt tccagagcca gatgccatca aggagtccct gctgtacttg
     2821 tacatggaca ggacctggat gccctatata ttttctctgt ccttggaggc tctgaaatgt
     2881 ttccgcatca ggaacaatga gaagatgctg agtgacagcc acggcgtgga gaccatccgg
     2941 gacatcctgc cagacaccag ccttgggggc ccatccttct tcaaaatcat cacggccaag
     3001 gctgtcctga agctgcaggc cggaaacgcc gaggaagccg ccctgtggag ggatctggtc
     3061 cgcaaagtcc tggcatccta cttggagaca gccgaggagg cggtgaccct gggcgggagc
     3121 ctggatgaaa actgtcagga ggtgctgaaa tttgccaccc gggagaatgg cttcctgctg
     3181 cagtacctgg tggctatccc catggagaaa ggccttgact cccaaggctg cttctgcgca
     3241 ggctgctccc ggcagatcgg cttctccttt gtacgaccca agctctgtgc cttctctggc
     3301 ctctattact gtgacatctg ccaccaagac gatgcctcag tgattccggc caggatcatc
     3361 cacaactggg acctcaccaa gcgcccgatc tgcaggcagg ccctgaagtt tctgacacag
     3421 atccgggccc agcccctcat caacctgcag atggtgaacg cgtctctgta cgagcatgtg
     3481 gagcggatgc acctcattgg gaggagacgg gagcagctga agctcctggg ggattacctg
     3541 ggcctgtgcc ggagtggcgc cctgaaggag ctcagcaaga ggctcaacca caggaattat
     3601 ctcttggaat ctccgcatag gttcagtgtt gctgacctcc aacagatcgc agacggggtg
     3661 tatgaaggat tcctcaaggc cctgattgaa tttgcctccc agcatgtcta ccactgcgac
     3721 ctgtgcaccc agcgcggctt catctgccag atctgccagc accacgacat catcttcccc
     3781 tttgagtttg acaccacagt caggtgtgcc gagtgcaaga ccgtcttcca ccagagctgc
     3841 caggctgtgg tgaagaaggg ctgcccccgc tgtgcccgcc ggcgcaagta ccaggaacag
     3901 aacattttcg cctgaggtac cgcgggcccg ggatccaccg gatctagata actgatcata
     3961 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     4021 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     4081 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4141 cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt
     4201 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     4261 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     4321 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     4381 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     4441 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     4501 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     4561 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     4621 taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4681 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4741 ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg
     4801 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4861 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     4921 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     4981 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     5041 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     5101 ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc
     5161 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     5221 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     5281 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     5341 tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     5401 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     5461 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     5521 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     5581 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     5641 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat
     5701 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     5761 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     5821 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     5881 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     5941 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     6001 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     6061 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     6121 ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat accggaagga
     6181 acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt
     6241 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
     6301 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
     6361 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca
     6421 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6481 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6541 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6601 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6661 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6721 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6781 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6841 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6901 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6961 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7021 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7081 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7141 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7201 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     7261 accgccatgc attagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     7321 tatggagttc cg
//
