LOCUS       sequence-470296-        6148 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression of HLA-A*24:02 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 6148)
  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 6148)
  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: "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-A*24:02
FEATURES             Location/Qualifiers
     source          1..6148
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(66..113)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     109..127
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(180..197)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1129..1151
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1166..1190
                     /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)."
     primer_bind     complement(1190..1209)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1191..1209)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1293..1312)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1518..1595
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1596..2678
                     /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     complement(1615..1634)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3487..3678
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3672..3694)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3780..3919
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3835..3854
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3934..3951)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4105..4693)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4185..4204)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4804..4823)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4815..5630
                     /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             4815..5630
                     /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     complement(4888..4907)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5722..6148,1..29))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5859..5880)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6071..6090
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca atccggatat agttcctcct
       61 ttcagcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc tagttattgc
      121 tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt tgttagcagc cggatctcag
      181 tggtggtggt ggtggtgctc gagtgcggcc gcaagctttt aacgatgatt ccacaccatt
      241 ttctgtgcat ccagaatatg atgcagggat cagggctggg aagatggctc ccatctcagg
      301 gtgaggggct tgggcagacc ctcatgctgc acatggcagg tgtatctctg ctcctctcca
      361 gaaggtacca ccacagctgc ccacttctgg aaggttccat cccctgcagg cctggtctcc
      421 acaagctccg tgtcctgggt ctggtcctcc ccatcccgct gccaggtcag tgtgatctcc
      481 gcagggtaga agcccagggc ccagcatctc agagtggcct catggtcaga gatggggtgg
      541 tgggtcatat gtgtcttggg ggggtccgtg cgctgcagcg tctccttccc gttctccagg
      601 tatctgcgga gcccgtccac gcacgtgccc tccaggtagg ctctctgctg ctccgccaca
      661 tgggccgcct cccacttgcg cttggtgatc tgagccgcca tgtccgccgc ggtccaagag
      721 cgcaggtcct ctttcagggc gatgtaatcc ttgcagtcgt aggcgtactg gtggtacccg
      781 cggaggaagc gcccgtccga ccccacgtcg cagccaaaca tcatctggag ggtgtgagaa
      841 ccggcctcgc tctggttgta gtagcggagc gcgatccgca ggttctctcg gtcagtctgt
      901 gagtgggcct tcactttccc tgtctcctcg tcccaatact ccggcccctc ctgctctatc
      961 cacggcgccc gcggctccat cctctggctc gcggcgtcgc tgtcgaaccg cacgaactgc
     1021 gtgtcgtcca cgtagcccac ggcgatgaag cggggctccc cgcggccggg ccgggacacg
     1081 gatgtggaga aatatctcat agaatgagaa ccgctagcca tatgtatatc tccttcttaa
     1141 agttaaacaa aattatttct agaggggaat tgttatccgc tcacaattcc cctatagtga
     1201 gtcgtattaa tttcgcggga tcgagatctc gatcctctac gccggacgca tcgtggccgg
     1261 catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga
     1321 agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg
     1381 ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc
     1441 ggtgttcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg
     1501 agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat
     1561 agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg
     1621 tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc
     1681 acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc
     1741 ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct
     1801 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     1861 aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     1921 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     1981 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     2041 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac
     2101 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     2161 taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc
     2221 aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa
     2281 ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
     2341 tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct
     2401 cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca
     2461 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     2521 agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca
     2581 ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     2641 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt
     2701 tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc
     2761 agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt
     2821 atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc
     2881 tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc
     2941 ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt
     3001 atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg
     3061 ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg
     3121 tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt
     3181 ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg
     3241 atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag
     3301 cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt
     3361 ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc
     3421 atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag
     3481 gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag
     3541 acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt
     3601 gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat
     3661 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     3721 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     3781 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat
     3841 cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag
     3901 gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3961 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4021 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4081 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4141 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4201 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4261 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4321 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4381 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4441 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4501 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     4561 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4621 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4681 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4741 aaactcacgt taagggattt tggtcatgaa caataaaact gtctgcttac ataaacagta
     4801 atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctctagg ccgcgattaa
     4861 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     4921 caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac
     4981 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
     5041 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
     5101 tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt
     5161 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
     5221 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
     5281 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     5341 aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc
     5401 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     5461 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     5521 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     5581 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa gaattaattc
     5641 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5701 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     5761 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     5821 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     5881 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     5941 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     6001 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     6061 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     6121 tcacgctgcg cgtaaccacc acacccgc
//
