LOCUS       sequence-68124-e        7458 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MBP myocilin olfactomedin domain fusion protein with a TEV
            protease recognition sequence and the mutation G252R.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7458)
  AUTHORS   Burns JN, Turnage KC, Walker CA, Lieberman RL
  TITLE     The stability of myocilin olfactomedin domain variants provides new 
            insight into glaucoma as a protein misfolding disorder.
  JOURNAL   Biochemistry. 2011 Jul 5;50(26):5824-33. doi: 10.1021/bi200231x. 
            Epub 2011 Jun 9.
  PUBMED    21612213
REFERENCE   2  (bases 1 to 7458)
  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: "10.1021/bi200231x";
            journalName: "Biochemistry"; date: "2011-07-5- 5"; volume: "50"; 
            issue: "26"; pages: "5824-33"
COMMENT     Sequence Label: pmalc4x_G252R_TEV
FEATURES             Location/Qualifiers
     source          1..7458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(855..872)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(855..871)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(864..886)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1050..1067)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1220..1306
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1398..1425
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1444..1535
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1536..1604,1605..2396)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1754..1773)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(2438..2951)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2633..2654)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2845..2864
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      3062..3650
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3551..3570
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3804..3821
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3836..3978
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3904..3923)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4064..4086
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4080..4271)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        4767..4844
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4845..5927
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4864..4883)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6170..6175,6176..6191,6192..6198)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    6206..6222
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     6211..6233
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             6292..7392
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     7366..7389
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             7399..7419
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
ORIGIN
        1 ggagacaccg gatgtggaga actagtttgg gtaagagagc ctctcacgct gagaacagca
       61 gaaacaatta ctggcaagta tggtgtgtgg atgcgagacc ccaagcccac ctacccctac
      121 acccaggaga ccacgtggag aatcgacaca gttggcacgg atgtccgcca ggtttttgag
      181 tatgacctca tcagccagtt tatgcagggc tacccttcta aggttcacat actgcctagg
      241 ccactggaaa gcacgggtgc tgtggtgtac tcggggagcc tctatttcca gggcgctgag
      301 tccagaactg tcataagata tgagctgaat accgagacag tgaaggctga gaaggaaatc
      361 cctggagctg gctaccacgg acagttcccg tattcttggg gtggctacac ggacattgac
      421 ttggctgtgg atgaagcagg cctctgggtc atttacagca ccgatgaggc caaaggtgcc
      481 attgtcctct ccaaactgaa cccagagaat ctggaactcg aacaaacctg ggagacaaac
      541 atccgtaagc agtcagtcgc caatgccttc atcatctgtg gcaccttgta caccgtcagc
      601 agctacacct cagcagatgc taccgtcaac tttgcttatg acacaggcac aggtatcagc
      661 aagaccctga ccatcccatt caagaaccgc tataagtaca gcagcatgat tgactacaac
      721 cccctggaga agaagctctt tgcctgggac aacttgaaca tggtcactta tgacatcaag
      781 ctttccaaga tgtgataagg ctctaactct cctgaattcg gatcctctag agtcgacctg
      841 caggcaagct tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
      901 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
      961 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcagcttggc
     1021 tgttttggcg gatgagataa gattttcagc ctgatacaga ttaaatcaga acgcagaagc
     1081 ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg
     1141 ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga
     1201 gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     1261 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga
     1321 tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc
     1381 caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac
     1441 tcttttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     1501 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     1561 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     1621 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     1681 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgtttccca atgatgagca
     1741 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac
     1801 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     1861 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     1921 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     1981 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     2041 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     2101 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     2161 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     2221 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     2281 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     2341 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     2401 cagaccaagt ttactcatat atactttaga ttgatttacc ccggttgata atcagaaaag
     2461 ccccaaaaac aggaagattg tataagcaaa tatttaaatt gtaaacgtta atattttgtt
     2521 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     2581 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     2641 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     2701 tcagggcgat ggcccactac gtgaaccatc acccaaatca agttttttgg ggtcgaggtg
     2761 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     2821 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     2881 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     2941 acagggcgcg taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     3001 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3061 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3121 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3181 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     3241 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3301 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3361 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3421 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3481 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3541 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3601 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3661 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     3721 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3781 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     3841 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatat ggtgcactct
     3901 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     3961 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4021 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4081 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     4141 tggtcgtgca gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     4201 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     4261 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     4321 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     4381 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     4441 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     4501 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     4561 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     4621 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     4681 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc
     4741 caggacccaa cgctgcccga aattccgaca ccatcgaatg gtgcaaaacc tttcgcggta
     4801 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     4861 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     4921 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     4981 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     5041 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     5101 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     5161 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     5221 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     5281 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     5341 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     5401 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     5461 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     5521 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     5581 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     5641 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     5701 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     5761 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     5821 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     5881 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     5941 aatgtaagtt agctcactca ttaggcacaa ttctcatgtt tgacagctta tcatcgactg
     6001 cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta tggctgtgca
     6061 ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt
     6121 ttttgcgccg acatcataac ggttctggca aatattctga aatgagctgt tgacaattaa
     6181 tcatcggctc gtataatgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     6241 cagtccgttt aggtgttttc acgagcactt caccaacaag gaccatagca tatgaaaatc
     6301 gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa
     6361 gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa
     6421 ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg
     6481 gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac
     6541 aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag
     6601 ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg
     6661 ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa
     6721 ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct
     6781 gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc
     6841 gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa
     6901 cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca
     6961 gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat
     7021 ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg
     7081 agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac
     7141 tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta
     7201 gcgctgaagt cttacgagga agagttggtg aaagatccgc ggattgccgc cactatggaa
     7261 aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc
     7321 gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa
     7381 gacgcgcaga ctaattcgga gaatctttat tttcagagct tgaaggagag cccatctggc
     7441 tatctcagga gtggagag
//
