LOCUS       sequence-355125-        6130 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 6130)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 6130)
  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            "(unpublished)"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: pTCF176 HLA-A*02:01 (CD8 mut)
FEATURES             Location/Qualifiers
     source          1..6130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     68..87
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        68..86
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    87..111
                     /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             126..148
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1062..1079
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1132..1150)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1146..1193
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1230..1685
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1317..1336)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1527..1548
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1777..2592)
                     /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(1777..2592)
                     /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     2500..2519
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2584..2603
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2714..3302
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3203..3222
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3456..3473
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3488..3627
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3553..3572)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3713..3735
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3729..3920)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4729..5811)
                     /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     5773..5792
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5812..5889)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6095..6114
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
       61 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
      121 ataattttgt ttaactttaa gaaggagata tacatatggg cagtcatagc atgcgctact
      181 tcttcaccag cgtgagtcgt cctggtcgtg gcgaaccgcg cttcatcgcc gttggttacg
      241 ttgacgacac ccaattcgtg cgcttcgata gcgacgcagc cagtcagcgt atggaaccgc
      301 gcgcaccgtg gatcgagcag gaaggtccgg agtactggga cggcgaaacc cgcaaggtta
      361 aagcccacag ccagacccat cgtgtggatc tgggcaccct gcgcggctac tacaaccaga
      421 gtgaggccgg cagccacacc gtgcaacgca tgtatggctg tgacgtgggt agcgactggc
      481 gcttcctgcg tggctaccat cagtacgcct acgacggcaa ggattacatc gccctgaaag
      541 aggacttacg cagctggaca gcagcagaca tggccgccca gaccaccaag cacaaatggg
      601 aagcagccca tgtggccgag cagctgcgtg catatctgga gggtacctgc gttgagtggc
      661 tgcgccgcta cctggagaac ggtaaggaga ccctgcagcg caccgacgca ccgaaaaccc
      721 acatgaccca ccacgccgtt agcgatcacg aagccacact gcgctgctgg gccctgagtt
      781 tctacccggc cgagatcacc ctgacctggc aacgtgacgg cgaggaccag acccagaagg
      841 ccgaactggt tgagacacgc cctgccggtg acggcacatt tcagaaatgg gccgccgttg
      901 tggtgccgag cggtcaagag cagcgctaca cctgccacgt gcagcatgaa ggcctgccga
      961 agcctttaac cctgcgctgg gagccgggat ccctgcatca tattctggat gcacagaaaa
     1021 tggtgtggaa tcatcgttaa aagcttgcgg ccgcactcga gcaccaccac caccaccact
     1081 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     1141 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     1201 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     1261 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1321 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     1381 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     1441 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     1501 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     1561 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     1621 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     1681 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1741 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     1801 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     1861 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     1921 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     1981 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     2041 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     2101 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     2161 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     2221 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     2281 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     2341 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     2401 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     2461 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     2521 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     2581 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     2641 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     2701 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2761 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2821 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     2881 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2941 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3001 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3061 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3121 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3181 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3241 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3301 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3361 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3421 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3481 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     3541 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     3601 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     3661 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3721 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     3781 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     3841 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     3901 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     3961 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     4021 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     4081 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     4141 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     4201 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     4261 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     4321 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     4381 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     4441 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     4501 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     4561 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     4621 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     4681 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     4741 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4801 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     4861 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     4921 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     4981 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     5041 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     5101 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     5161 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     5221 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     5281 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     5341 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     5401 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     5461 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     5521 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     5581 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     5641 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     5701 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     5761 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     5821 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     5881 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     5941 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     6001 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     6061 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     6121 caaccgcacc
//
