LOCUS       sequence-68116-e        7458 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses MBP myocilin olfactomedin domain fusion protein with a TEV
            protease recognition sequence and the mutation K398R.
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 Apr 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_K398R_TEV
FEATURES             Location/Qualifiers
     source          1..7458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..80
                     /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             81..1163
                     /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(100..119)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1406..1411,1412..1427,1428..1434)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1442..1458
                     /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     1447..1469
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1528..2628
                     /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     2602..2625
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2635..2655
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(3549..3566)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3549..3565)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3558..3580)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3744..3761)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3914..4000
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4092..4119
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4138..4229
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4230..4298,4299..5090)
                     /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(4448..4467)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5132..5645)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5327..5348)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5539..5558
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5756..6344
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6245..6264
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6498..6515
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6530..6672
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6598..6617)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6758..6780
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6774..6965)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
       61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
      901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
      961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1501 gcacttcacc aacaaggacc atagcatatg aaaatcgaag aaggtaaact ggtaatctgg
     1561 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
     1621 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
     1681 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
     1741 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
     1801 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
     1861 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa
     1921 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
     1981 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
     2041 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
     2101 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
     2161 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
     2221 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
     2281 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
     2341 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
     2401 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     2461 ttggtgaaag atccgcggat tgccgccact atggaaaacg cccagaaagg tgaaatcatg
     2521 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     2581 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcggagaat
     2641 ctttattttc agagcttgaa ggagagccca tctggctatc tcaggagtgg agagggagac
     2701 accggatgtg gagaactagt ttgggtagga gagcctctca cgctgagaac agcagaaaca
     2761 attactggca agtatggtgt gtggatgcga gaccccaagc ccacctaccc ctacacccag
     2821 gagaccacgt ggagaatcga cacagttggc acggatgtcc gccaggtttt tgagtatgac
     2881 ctcatcagcc agtttatgca gggctaccct tctaaggttc acatactgcc taggccactg
     2941 gaaagcacgg gtgctgtggt gtactcgggg agcctctatt tccagggcgc tgagtccaga
     3001 actgtcataa gatatgagct gaataccgag acagtgaagg ctgagaagga aatccctgga
     3061 gctggctacc acggacagtt cccgtattct tggggtggct acacggacat tgacttggct
     3121 gtggatgaag caggcctctg ggtcatttac agcaccgatg aggccagagg tgccattgtc
     3181 ctctccaaac tgaacccaga gaatctggaa ctcgaacaaa cctgggagac aaacatccgt
     3241 aagcagtcag tcgccaatgc cttcatcatc tgtggcacct tgtacaccgt cagcagctac
     3301 acctcagcag atgctaccgt caactttgct tatgacacag gcacaggtat cagcaagacc
     3361 ctgaccatcc cattcaagaa ccgctataag tacagcagca tgattgacta caaccccctg
     3421 gagaagaagc tctttgcctg ggacaacttg aacatggtca cttatgacat caagctttcc
     3481 aagatgtgat aaggctctaa ctctcctgaa ttcggatcct ctagagtcga cctgcaggca
     3541 agcttggcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     3601 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     3661 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcagct tggctgtttt
     3721 ggcggatgag ataagatttt cagcctgata cagattaaat cagaacgcag aagcggtctg
     3781 ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac
     3841 tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg
     3901 aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat
     3961 ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa
     4021 cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca
     4081 tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactctttt
     4141 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4201 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     4261 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     4321 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     4381 gcggtaagat ccttgagagt tttcgccccg aagaacgttt cccaatgatg agcactttta
     4441 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
     4501 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     4561 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     4621 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     4681 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     4741 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     4801 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     4861 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     4921 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     4981 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5041 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     5101 aagtttactc atatatactt tagattgatt taccccggtt gataatcaga aaagccccaa
     5161 aaacaggaag attgtataag caaatattta aattgtaaac gttaatattt tgttaaaatt
     5221 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     5281 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     5341 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     5401 cgatggccca ctacgtgaac catcacccaa atcaagtttt ttggggtcga ggtgccgtaa
     5461 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     5521 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     5581 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     5641 cgcgtaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5701 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5761 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5821 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5881 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     5941 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6001 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6061 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6121 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6181 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6241 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6301 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6361 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6421 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     6481 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     6541 attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac
     6601 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     6661 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     6721 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     6781 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     6841 tgcagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     6901 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     6961 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     7021 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     7081 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     7141 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     7201 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     7261 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     7321 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     7381 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac
     7441 ccaacgctgc ccgaaatt
//
