LOCUS       sequence-426781-        5704 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression of human beta-2-microglobulin containing a 
            cysteine mutation for disulfide linkage to mutant HLA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5704)
  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 5704)
  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 Beta2-microglobulin
FEATURES             Location/Qualifiers
     source          1..5704
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    733..872
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(798..817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     958..980
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(974..1165)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3017..3036
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3056..3133)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3339..3358
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3442..3461
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3442..3460
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3461..3485
                     /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             3500..3522
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3878..3895
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3948..3966)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3962..4009
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4046..4501
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4133..4152)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4343..4364
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4527..4631
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4632..4700,4701..5492)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4850..4869)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5663..5704,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      781 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
      841 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
      901 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
      961 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     1021 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     1081 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     1141 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     1201 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     1261 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     1321 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     1381 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     1441 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     1501 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     1561 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     1621 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     1681 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     1741 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     1801 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     1861 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     1921 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     1981 ccgctttcca gtcgggaaac tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2041 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     2101 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     2161 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     2221 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     2281 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     2341 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     2401 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     2461 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     2521 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     2581 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     2641 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     2701 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     2761 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     2821 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     2881 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     2941 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     3001 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     3061 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     3121 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     3181 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     3241 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     3301 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     3361 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     3421 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     3481 attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat gatccagcgt
     3541 actccaaaga ttcaggttta ctcacgtcat ccagcagaga atggaaagtc aaatttcctg
     3601 aattgctatg tgtctgggtt ttgtccatcc gacattgaag ttgacttact gaagaatgga
     3661 gagagaattg aaaaagtgga gcattcagac ttatctttca gcaaggactg gtctttctat
     3721 ctcttgtact acactgaatt cacccccact gaaaaagatg agtatgcctg ccgtgtgaac
     3781 catgtgactt tgtcacagcc caagatagtt aagtgggatc gagacatgta aggatccgaa
     3841 ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactgaga
     3901 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     3961 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     4021 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4081 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     4141 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4201 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4261 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4321 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     4381 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     4441 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     4501 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4561 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4621 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4681 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4741 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4801 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     4861 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     4921 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     4981 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5041 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     5101 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     5161 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     5221 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     5281 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5341 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     5401 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     5461 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     5521 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5581 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5641 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5701 aaac
//
