LOCUS       sequence-355080-        6142 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression of soluble MHC protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6142)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 6142)
  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"; journalName: 
            "(unpublished)"
COMMENT     Sequence Label: pTCF194 Mamu-E*02:04
FEATURES             Location/Qualifiers
     source          1..6142
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(396..1478)
                     /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     1440..1459
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1479..1556)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1762..1781
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1865..1884
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1865..1883
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1884..1908
                     /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             1923..1945
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2871..2888
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2941..2959)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2955..3002
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3039..3494
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3126..3145)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3336..3357
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3586..4401)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3586..4401)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4309..4328
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4393..4412
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4523..5111
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5012..5031
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5265..5282
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5297..5436
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5362..5381)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5522..5544
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5538..5729)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
       61 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
      121 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
      181 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
      241 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
      301 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
      361 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
      421 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
      481 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
      541 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
      601 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
      661 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
      721 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
      781 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
      841 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
      901 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
      961 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     1021 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     1081 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     1141 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     1201 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     1261 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     1321 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     1381 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     1441 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     1501 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     1561 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     1621 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     1681 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     1741 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     1801 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     1861 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     1921 attttgttta actttaagaa ggagatatac atatgggctc gcattccctg aaatatttcc
     1981 atacgagtgt cagtcgccca ggcggaggcg gccctcgctt tatctccgtc ggatatgtgg
     2041 atgataccca attcgtccgt tatgactccg acgcggcctc gccgcgcatg gaaccgcgtg
     2101 ctctgtggat gaaacaggag ggtcctgaat attgggacca agaaacacgt tcagcgcgtg
     2161 caacggcgca gacctttcgc gtgaatcttg aaaccctccg tggctattac aatcagagtg
     2221 aggcaggctc tcatacctta cagtggatgc atggctgcga ccttggtcca gacggccgct
     2281 ttttacgcgg atatgaacag tttgcctacg atggcaaaga ttatctgaca cttaacgaag
     2341 acctgcgctc ctggtctgcg gtggataccg ctgcgcaaat ttcggagcaa aaaagcaacg
     2401 acggatctga ggcggaacat cagcgcgctt atctggagga cacttgtgtt gaatggttac
     2461 gccgctacct ggaaaatggt aaagaaaccc ttcaacgtag cgaaagccca aaaacgcatg
     2521 tgactcacca cccggtctcg gaccacgaag cgactctgcg ttgctgggca cttggctttt
     2581 atccggcaga aatcacggtg acctggcagc gggacggcga agaccagacc caagatacag
     2641 aactggtcga aacacgccct gccggcgacg gcacgttcca gaaatgggcg gcagttgtgg
     2701 tgccgtccgg tgaagaacag cgttacacct gccatgtaca acacgaaggt ctgccggaac
     2761 cgttaacgct tcggtgggaa cccagcagcc agtcaggatc cctgcatcat attctggatg
     2821 cacagaaaat ggtgtggaat catcgttaaa agcttgcggc cgcactcgag caccaccacc
     2881 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     2941 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     3001 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     3061 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3121 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3181 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3241 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3301 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3361 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3421 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     3481 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3541 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     3601 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     3661 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     3721 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     3781 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     3841 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     3901 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     3961 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     4021 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     4081 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     4141 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     4201 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     4261 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     4321 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     4381 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     4441 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4501 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4561 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4621 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     4681 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4741 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4801 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4861 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4921 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4981 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5041 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5101 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5161 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     5221 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     5281 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     5341 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     5401 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     5461 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5521 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     5581 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     5641 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     5701 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     5761 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     5821 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     5881 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     5941 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     6001 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     6061 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     6121 ttctgctaac cagtaaggca ac
//
