LOCUS       sequence-355842-        6139 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 6139)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 6139)
  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            "(unpublished)"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: pTCF120 H-2D(b)
FEATURES             Location/Qualifiers
     source          1..6139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             925..942
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(995..1013)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1009..1056
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1093..1548
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1180..1199)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1390..1411
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1640..2455)
                     /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(1640..2455)
                     /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     2363..2382
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2447..2466
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2577..3165
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3066..3085
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3319..3336
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3351..3490
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3416..3435)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3576..3598
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3592..3783)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4592..5674)
                     /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     5636..5655
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5675..5752)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5958..5977
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6061..6080
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6061..6079
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6080..6104
                     /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             join(6119..6139,1..2)
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gatatacata tgggtccgca tagcatgcgt tattttgaaa ccgcagttag ccgtccgggt
       61 ctggaagaac cgcgttatat tagcgttggt tatgtggata acaaagaatt cgtgcgtttt
      121 gattccgatg cagaaaatcc gcgttatgaa ccgcgtgcac cgtggatgga acaagaaggt
      181 ccggaatatt gggaacgtga aacccagaaa gcaaaaggtc aagaacagtg gtttcgtgtt
      241 agcctgcgta atctgctggg ctattataac cagagtgccg gtggtagcca taccctgcag
      301 cagatgagcg gttgtgatct gggtagcgat tggcgtctgc tgcgtggtta tctgcagttt
      361 gcctatgaag gtcgtgatta tattgccctg aatgaggatc tgaaaacctg gaccgcagca
      421 gatatggcag cacagattac ccgtcgtaaa tgggaacaga gcggtgcagc agaacactat
      481 aaagcatatc tggaaggtga atgtgttgaa tggctgcatc gttatctgaa aaatggtaat
      541 gcaaccctgc tgcgtaccga tagcccgaaa gcacatgtta cccatcatcc gcgtagcaaa
      601 ggtgaagtta ccctgcgttg ttgggcactg ggtttttatc cggcagatat taccctgacc
      661 tggcagctga atggtgaaga actgacccag gatatggaac tggttgaaac ccgtcctgcc
      721 ggtgatggca cctttcagaa atgggcaagc gttgttgttc cgctgggtaa agaacagaat
      781 tatacctgtc gtgtgtatca tgaaggtctg ccggaaccgc tgaccctgcg ctgggaaccg
      841 cctccgagtg gatccctgca tcatattctg gatgcacaga aaatggtgtg gaatcatcgt
      901 taaaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac
      961 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     1021 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     1081 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1141 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1201 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1261 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1321 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1381 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1441 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1501 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     1561 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     1621 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     1681 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     1741 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     1801 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     1861 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     1921 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     1981 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     2041 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     2101 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     2161 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     2221 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     2281 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     2341 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     2401 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     2461 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     2521 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2581 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2641 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2701 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     2761 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2821 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     2881 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     2941 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3001 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3061 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3121 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3181 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3241 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3301 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3361 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     3421 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     3481 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3541 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     3601 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     3661 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     3721 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     3781 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     3841 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     3901 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     3961 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     4021 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     4081 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     4141 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     4201 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     4261 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     4321 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     4381 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     4441 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     4501 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     4561 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4621 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4681 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     4741 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     4801 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     4861 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     4921 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     4981 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     5041 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     5101 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     5161 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     5221 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     5281 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     5341 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     5401 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     5461 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     5521 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     5581 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     5641 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     5701 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     5761 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     5821 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     5881 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     5941 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     6001 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     6061 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
     6121 tgtttaactt taagaagga
//
