LOCUS       sequence-311578-        5994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of MIF Y99F in E. coli for protein production.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5994)
  TITLE     Lolis Lab Plasmids
REFERENCE   2  (bases 1 to 5994)
  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"
COMMENT     Sequence Label: pET11b-MIF Y99F
FEATURES             Location/Qualifiers
     source          1..5994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(495..513)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      509..556
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(731..736,737..753,754..759))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(786..804)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        872..976
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(977..1045,1046..1837)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1195..1214)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2008..2596
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2497..2516
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2750..2767
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2782..2921
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2847..2866)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3007..3029
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3023..3214)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4526..5608)
                     /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
                     EACKAAVHNLLAQRVSGLIINYPLYDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5570..5589
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5609..5686)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5892..5911
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gccgatgttc atcgtaaaca ccaacgtgcc
      121 ccgcgcctcc gtgccggacg ggttcctctc cgagctcacc cagcagctgg cgcaggccac
      181 cggcaagccc ccccagtaca tcgcggtgca cgtggtcccg gaccagctca tggccttcgg
      241 cggctccagc gagccgtgcg cgctctgcag cctgcacagc atcggcaaga tcggcggcgc
      301 gcagaaccgc tcctacagca agctgctgtg cggcctgctg gccgagcgcc tgcgcatcag
      361 cccggacagg gtctacatca actattttga catgaacgcg gccaatgtgg gctggaacaa
      421 ctccaccttc gcctaagagc cgcaggatcc ggctgctaac aaagcccgaa aggaagctga
      481 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
      541 cttgaggggt tttttgctga aaggaggaac tatatccgga tatcccgcaa gaggcccggc
      601 agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc gaggatgacg
      661 atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt taactgtgat
      721 aaactaccgc attaaagctt atcgatgata agctgtcaaa catgagaatt cttgaagacg
      781 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
      841 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
      901 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
      961 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     1021 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     1081 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     1141 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     1201 tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     1261 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     1321 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     1381 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     1441 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     1501 gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga
     1561 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     1621 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     1681 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     1741 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     1801 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     1861 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     1921 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     1981 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2041 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2101 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     2161 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2221 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2281 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2341 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2401 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     2461 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     2521 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     2581 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     2641 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     2701 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     2761 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     2821 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     2881 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     2941 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3001 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     3061 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     3121 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     3181 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     3241 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     3301 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     3361 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     3421 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     3481 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     3541 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     3601 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     3661 gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc
     3721 tgctggagat ggcggacgcg atggatatgt tctgccaagg gttggtttgc gcattcacag
     3781 ttctccgcaa gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc
     3841 gccggcttcc attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag
     3901 gcagacaagg tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga
     3961 ggcggcataa atcgccgtga cgatcagcgg tccaatgatc gaagttaggc tggtaagagc
     4021 cgcgagcgat ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc
     4081 ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat
     4141 ccagcctcgc gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc
     4201 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     4261 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     4321 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     4381 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     4441 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     4501 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4561 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     4621 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     4681 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     4741 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     4801 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     4861 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     4921 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     4981 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     5041 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     5101 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5161 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     5221 catacagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     5281 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     5341 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     5401 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     5461 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     5521 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     5581 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     5641 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     5701 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     5761 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     5821 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     5881 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     5941 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaat
//
