LOCUS       sequence-68122-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 I499F.
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_I499F_TEV
FEATURES             Location/Qualifiers
     source          1..7458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     139..156
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    171..313
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(239..258)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     399..421
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(415..606)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        1102..1179
                     /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             1180..2262
                     /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(1199..1218)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(2505..2510,2511..2526,2527..2533)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2541..2557
                     /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     2546..2568
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             2627..3727
                     /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     3701..3724
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3734..3754
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(4648..4665)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4648..4664)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4657..4679)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4843..4860)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5013..5099
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5191..5218
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        5237..5328
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5329..5397,5398..6189)
                     /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(5547..5566)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6231..6744)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6426..6447)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6638..6657
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      6855..7443
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7344..7363
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
       61 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
      121 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
      181 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta
      241 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
      301 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
      361 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
      421 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
      481 gtgcagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
      541 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
      601 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
      661 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
      721 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
      781 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
      841 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
      901 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
      961 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     1021 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggccagga
     1081 cccaacgctg cccgaaattc cgacaccatc gaatggtgca aaacctttcg cggtatggca
     1141 tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac
     1201 gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc
     1261 agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac
     1321 attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc
     1381 acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc
     1441 gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt
     1501 aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg
     1561 ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt
     1621 cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg
     1681 cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc
     1741 ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc
     1801 aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa
     1861 caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat
     1921 cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat
     1981 atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc
     2041 accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc
     2101 tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa
     2161 accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     2221 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     2281 aagttagctc actcattagg cacaattctc atgtttgaca gcttatcatc gactgcacgg
     2341 tgcaccaatg cttctggcgt caggcagcca tcggaagctg tggtatggct gtgcaggtcg
     2401 taaatcactg cataattcgt gtcgctcaag gcgcactccc gttctggata atgttttttg
     2461 cgccgacatc ataacggttc tggcaaatat tctgaaatga gctgttgaca attaatcatc
     2521 ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagccagtc
     2581 cgtttaggtg ttttcacgag cacttcacca acaaggacca tagcatatga aaatcgaaga
     2641 aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg
     2701 taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga
     2761 agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca
     2821 cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc
     2881 gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat
     2941 tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa
     3001 cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa
     3061 gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga
     3121 cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga
     3181 taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat
     3241 gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat
     3301 gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt
     3361 aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc
     3421 aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg aaaactatct
     3481 gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct
     3541 gaagtcttac gaggaagagt tggtgaaaga tccgcggatt gccgccacta tggaaaacgc
     3601 ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg
     3661 tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc
     3721 gcagactaat tcggagaatc tttattttca gagcttgaag gagagcccat ctggctatct
     3781 caggagtgga gagggagaca ccggatgtgg agaactagtt tgggtaggag agcctctcac
     3841 gctgagaaca gcagaaacaa ttactggcaa gtatggtgtg tggatgcgag accccaagcc
     3901 cacctacccc tacacccagg agaccacgtg gagaatcgac acagttggca cggatgtccg
     3961 ccaggttttt gagtatgacc tcatcagcca gtttatgcag ggctaccctt ctaaggttca
     4021 catactgcct aggccactgg aaagcacggg tgctgtggtg tactcgggga gcctctattt
     4081 ccagggcgct gagtccagaa ctgtcataag atatgagctg aataccgaga cagtgaaggc
     4141 tgagaaggaa atccctggag ctggctacca cggacagttc ccgtattctt ggggtggcta
     4201 cacggacatt gacttggctg tggatgaagc aggcctctgg gtcatttaca gcaccgatga
     4261 ggccaaaggt gccattgtcc tctccaaact gaacccagag aatctggaac tcgaacaaac
     4321 ctgggagaca aacatccgta agcagtcagt cgccaatgcc ttcatcatct gtggcacctt
     4381 gtacaccgtc agcagctaca cctcagcaga tgctaccgtc aactttgctt atgacacagg
     4441 cacaggtatc agcaagaccc tgaccatccc attcaagaac cgctataagt acagcagcat
     4501 gattgactac aaccccctgg agaagaagct ctttgcctgg gacaacttga acatggtcac
     4561 ttatgacttc aagctttcca agatgtgata aggctctaac tctcctgaat tcggatcctc
     4621 tagagtcgac ctgcaggcaa gcttggcact ggccgtcgtt ttacaacgtc gtgactggga
     4681 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     4741 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     4801 atggcagctt ggctgttttg gcggatgaga taagattttc agcctgatac agattaaatc
     4861 agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc
     4921 acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc
     4981 tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag
     5041 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc
     5101 cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc
     5161 cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg
     5221 cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5281 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5341 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5401 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5461 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttc
     5521 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc
     5581 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5641 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5701 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5761 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5821 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5881 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5941 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     6001 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     6061 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     6121 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6181 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt accccggttg
     6241 ataatcagaa aagccccaaa aacaggaaga ttgtataagc aaatatttaa attgtaaacg
     6301 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     6361 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     6421 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     6481 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcacccaaa tcaagttttt
     6541 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     6601 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     6661 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     6721 ttaatgcgcc gctacagggc gcgtaaaagg atctaggtga agatcctttt tgataatctc
     6781 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6841 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6901 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6961 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     7021 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     7081 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     7141 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     7201 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     7261 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     7321 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     7381 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     7441 aaaaacgcca gcaacgcg
//
