LOCUS       sequence-357353-        6152 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 6152)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 6152)
  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            "(unpublished)"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: pTCF10 HLA-B*08:01
FEATURES             Location/Qualifiers
     source          1..6152
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    40..64
                     /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             79..101
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1033..1050
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1103..1121)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1117..1164
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1201..1656
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1288..1307)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1498..1519
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1749..2564)
                     /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(1749..2564)
                     /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     2472..2491
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2556..2575
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2686..3274
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3175..3194
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3428..3445
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3460..3602
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3528..3547)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3688..3710
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3704..3895)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4704..5786)
                     /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     5748..5767
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5787..5864)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6070..6089
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
       61 ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat ggctagcggt
      121 tctcattcta tgagatattt cgacaccgcc atgtcccgac ccggccgcgg ggagccccgc
      181 ttcatctcag tgggctacgt ggacgacacg cagttcgtga ggttcgacag cgacgccgcg
      241 agtccgagag aggagccgcg ggcgccgtgg atagagcagg aggggccgga gtattgggac
      301 cggaacacac agatcttcaa gaccaacaca cagactgacc gagagagcct gcggaacctg
      361 cgcggctact acaaccagag cgaggccggg tctcacaccc tccagagcat gtacggctgc
      421 gacgtggggc cggacgggcg cctcctccgc gggcataacc agtacgccta cgacggcaag
      481 gattacatcg ccctgaacga ggacctgcgc tcctggaccg cggcggacac ggcggctcag
      541 atcacccagc gcaagtggga ggcggcccgt gtggcggagc aggacagagc ctacctggag
      601 ggcacgtgcg tggagtggct ccgcagatac ctggagaacg ggaaggacac gctggagcgc
      661 gcggaccccc caaagacaca cgtgacccac caccccatct ctgaccatga ggccaccctg
      721 aggtgctggg ccctgggctt ctaccctgcg gagatcacac tgacctggca gcgggatggc
      781 gaggaccaaa ctcaggacac tgagcttgtg gagaccagac cagcaggaga tagaaccttc
      841 cagaagtggg cagctgtggt ggtgccttct ggagaagagc agagatacac atgccatgta
      901 cagcatgagg ggctgccgaa gcccctcacc ctgagatggg agccgtcttc ccagtccgga
      961 tccctgcatc atattctgga tgcacagaaa atggtgtgga atcatcgtta aaagcttgcg
     1021 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     1081 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     1141 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     1201 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1261 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1321 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1381 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1441 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1501 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1561 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1621 acgcgaattt taacaaaata ttaacgttta caatttcagg tggcactttt cggggaaatg
     1681 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1741 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     1801 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     1861 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     1921 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     1981 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     2041 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     2101 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     2161 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     2221 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     2281 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     2341 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     2401 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     2461 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     2521 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     2581 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     2641 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2701 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2761 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2821 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2881 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2941 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3001 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3061 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3121 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3181 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3241 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3301 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3361 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3421 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3481 tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac aatctgctct
     3541 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     3601 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3661 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3721 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     3781 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     3841 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     3901 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     3961 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     4021 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     4081 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     4141 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     4201 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     4261 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     4321 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     4381 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     4441 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     4501 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     4561 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     4621 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     4681 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4741 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     4801 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     4861 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     4921 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     4981 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     5041 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     5101 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     5161 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     5221 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     5281 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5341 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5401 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     5461 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5521 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5581 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     5641 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     5701 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     5761 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     5821 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     5881 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     5941 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     6001 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     6061 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     6121 gccggccacg atgcgtccgg cgtagaggat cg
//
