LOCUS       sequence-357358-        6134 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 6134)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 6134)
  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            "(unpublished)"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..6134
                     /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(1778..2593)
                     /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(1778..2593)
                     /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     2501..2520
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2585..2604
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2715..3303
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3204..3223
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3457..3474
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3489..3631
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3557..3576)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3717..3739
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3733..3924)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4733..5815)
                     /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     5777..5796
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5816..5893)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6099..6118
                     /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 taacgtttac
     1681 aatttcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1741 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     1801 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     1861 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     1921 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     1981 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     2041 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     2101 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     2161 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     2221 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     2281 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     2341 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     2401 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     2461 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     2521 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     2581 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     2641 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     2701 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2761 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2821 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     2881 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     2941 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3001 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3061 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3121 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3181 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3241 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3301 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3361 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3421 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3481 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     3541 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     3601 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     3661 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3721 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     3781 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     3841 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     3901 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     3961 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     4021 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     4081 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     4141 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     4201 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     4261 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     4321 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     4381 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     4441 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     4501 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     4561 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     4621 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     4681 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     4741 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4801 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     4861 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     4921 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     4981 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     5041 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     5101 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     5161 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     5221 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     5281 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     5341 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     5401 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     5461 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     5521 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     5581 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     5641 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     5701 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     5761 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     5821 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     5881 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     5941 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     6001 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     6061 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     6121 ccagcaaccg cacc
//
