LOCUS       sequence-366471-        6142 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 6142)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 6142)
  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            "(unpublished)"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: pJA48 HLA-B*57:01
FEATURES             Location/Qualifiers
     source          1..6142
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(992..1010)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1006..1053
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1090..1545
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1177..1196)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1387..1408
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1637..2452)
                     /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(1637..2452)
                     /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     2360..2379
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2444..2463
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2574..3162
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3063..3082
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3316..3333
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3348..3487
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3413..3432)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3573..3595
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3589..3780)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4589..5671)
                     /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     5633..5652
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5672..5749)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5955..5974
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6058..6077
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6058..6076
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6077..6101
                     /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             6116..6138
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 catatgggtt ctcattctat gagatatttc tacaccgcca tgtcccggcc cggccgcggg
       61 gagccccgct tcatcgcagt gggctacgtg gacgacaccc agttcgtgag gttcgacagc
      121 gacgccgcga gtccgaggat ggcgccccgg gcgccatgga tagagcagga ggggccggag
      181 tattgggacg gggagacacg gaacatgaag gcctccgcgc agacttaccg agagaacctg
      241 cggatcgcgc tccgctacta caaccagagc gaggccgggt ctcacatcat ccaggtgatg
      301 tatggctgcg acgtggggcc ggacgggcgc ctcctccgcg ggcatgacca gtccgcctac
      361 gacggcaagg attacatcgc cctgaacgag gacctgagct cctggaccgc ggcggacacg
      421 gcggctcaga tcacccagcg caagtgggag gcggcccgtg tggcggagca gctgagagcc
      481 tacctggagg gcctgtgcgt ggagtggctc cgcagatacc tggagaacgg gaaggagacg
      541 ctgcagcgcg cggacccccc aaagacacat gtgacccacc accccatctc tgaccatgag
      601 gccaccctga ggtgctgggc cctgggcttc taccctgcgg agatcacact gacctggcag
      661 cgggatggcg aggaccaaac tcaggacacc gagcttgtgg agaccagacc agcaggagat
      721 agaaccttcc agaagtgggc agctgtggtg gtgccttctg gagaagagca gagatacaca
      781 tgccatgtac agcatgaggg gctgccaaag cccctcaccc tgagatggga gccatcttcc
      841 caatccggat ccctgcatca tattctggat gcacagaaaa tggtgtggaa tcatcgttaa
      901 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
      961 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     1021 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     1081 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1141 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     1201 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     1261 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     1321 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     1381 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     1441 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     1501 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     1561 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1621 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     1681 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     1741 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     1801 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     1861 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     1921 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     1981 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     2041 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     2101 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     2161 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     2221 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     2281 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     2341 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     2401 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     2461 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     2521 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2581 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2641 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2701 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     2761 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2821 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2881 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2941 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3001 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3061 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     3361 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     3421 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     3481 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     3541 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     3601 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     3661 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     3721 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     3781 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     3841 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     3901 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     3961 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     4021 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     4081 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     4141 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     4201 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     4261 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     4321 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     4381 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     4441 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     4501 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     4561 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4621 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4681 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     4741 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     4801 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     4861 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     4921 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     4981 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     5041 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     5101 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     5161 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     5221 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     5281 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     5341 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     5401 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     5461 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     5521 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     5581 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     5641 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     5701 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     5761 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     5821 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     5881 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     5941 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     6001 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     6061 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     6121 ttaactttaa gaaggagata ta
//
