LOCUS       sequence-479365-        6142 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  E. coli expression of BirA tagged HLA-B*07:02 containing a cysteine 
            mutation for disulfide linkage to mutant beta-2 microglobulin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6142)
  AUTHORS   Sun Y, Young MC, Woodward CH, Danon JN, Truong HV, Gupta S, Winters 
            TJ, Font-Burgada J, Burslem GM, Sgourakis NG
  TITLE     Universal open MHC-I molecules for rapid peptide loading and 
            enhanced complex stability across HLA allotypes.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Jun 20;120(25):e2304055120. doi: 
            10.1073/pnas.2304055120. Epub 2023 Jun 13.
  PUBMED    37310998
REFERENCE   2  (bases 1 to 6142)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 22, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2023-06-20"; volume: "120(25):e2304055120. 
            doi"; pages: " 10.1073/pnas.2304055120. Epub 2023 Jun 13"
COMMENT     Sequence Label: OPEN-HLA-B*07:02-BirA
FEATURES             Location/Qualifiers
     source          1..6142
                     /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)"
     CDS             1007..1024
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1077..1095)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1091..1138
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1175..1630
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1262..1281)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1472..1493
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1722..2537)
                     /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(1722..2537)
                     /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     2445..2464
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2529..2548
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2659..3247
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3148..3167
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3401..3418
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3433..3572
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3498..3517)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3658..3680
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3674..3865)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4674..5756)
                     /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     5718..5737
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5757..5834)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6040..6059
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gggttctcat tctatgagat atttctacac
      121 ctccgtgtcc cggcccggcc gcggggagcc ccgcttcatc tcagtgggct acgtggacga
      181 cacccagttc gtgaggttcg acagcgacgc cgcgagtccg agagaggagc cgcgggcgcc
      241 gtggatagag caggaggggc cggagtattg ggaccggaac acacagatct acaaggccca
      301 ggcacagact gaccgagaga gcctgcggaa cctgcgcggc tactacaacc agagcgaggc
      361 cgggtctcac accctccaga gcatgtacgg ctgcgacgtg gggccggacg ggcgcctcct
      421 ccgcgggcat gaccagtacg cctacgactg caaggattac atcgccctga acgaggacct
      481 gcgctcctgg accgccgcgg acacggcggc tcagatcacc cagcgcaagt gggaggcggc
      541 ccgtgaggcg gagcagcgga gagcctacct ggagggcgag tgcgtggagt ggctccgcag
      601 atacctggag aacgggaagg acaagctgga gcgcgctgac cccccaaaga cacacgtgac
      661 ccaccacccc atctctgacc atgaggccac cctgaggtgc tgggccctgg gtttctaccc
      721 tgcggagatc acactgacct ggcagcggga tggcgaggac caaactcagg acactgagct
      781 tgtggagacc agaccagcag gagatagaac cttccagaag tgggcagctg tggtggtgcc
      841 ttctggagaa gagcagagat acacatgcca tgtacagcat gaggggctgc cgaagcccct
      901 caccctgaga tgggagccgt cttcccagtc cggatccctg catcatattc tggatgcaca
      961 gaaaatggtg tggaatcatc gttaaaagct tgcggccgca ctcgagcacc accaccacca
     1021 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1081 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1141 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     1201 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1261 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1321 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1381 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1441 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1501 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1561 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     1621 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1681 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     1741 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     1801 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     1861 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     1921 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     1981 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2041 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     2101 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     2161 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2221 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2281 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     2341 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     2401 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     2461 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     2521 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     2581 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2641 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2701 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2761 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     2821 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     2881 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     2941 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3001 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3061 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3121 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3181 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3241 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3301 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3361 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3421 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3481 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     3541 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     3601 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3661 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     3721 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     3781 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     3841 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     3901 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     3961 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     4021 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     4081 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     4141 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     4201 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     4261 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4321 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     4381 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     4441 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     4501 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     4561 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     4621 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     4681 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4741 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     4801 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     4861 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     4921 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     4981 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     5041 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     5101 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     5161 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     5221 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     5281 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     5341 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5401 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     5461 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     5521 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     5581 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     5641 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     5701 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     5761 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     5821 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     5881 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     5941 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     6001 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     6061 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     6121 cgagatctcg atcccgcgaa at
//
