LOCUS       sequence-357357-        6152 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 6152)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 6152)
  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            "(unpublished)"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: pTCF5 HLA-A*01:01
FEATURES             Location/Qualifiers
     source          1..6152
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    43..67
                     /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             82..104
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1036..1053
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1106..1124)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1120..1167
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1204..1659
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1291..1310)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1501..1522
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1752..2567)
                     /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(1752..2567)
                     /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     2475..2494
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2559..2578
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2689..3277
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3178..3197
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3431..3448
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3463..3605
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3531..3550)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3691..3713
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3707..3898)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4707..5789)
                     /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     5751..5770
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5790..5867)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6073..6092
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
       61 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca tatggctagc
      121 ggttctcatt ctatgaggta tttcttcaca tccgtgtccc ggcccggccg cggggagccc
      181 cgcttcatcg ccgtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc
      241 gcgagccaga agatggagcc gcgggcgccg tggatagagc aggaggggcc ggagtattgg
      301 gaccaggaga cacggaatat gaaggcccac tcacagactg accgagcgaa cctggggacc
      361 ctgcgcggct actacaacca gagcgaggac ggttctcaca ccatccagat aatgtatggc
      421 tgcgacgtgg ggccggacgg gcgcttcctc cgcgggtacc ggcaggacgc ctacgacggc
      481 aaggattaca tcgccctgaa cgaggacctg cgctcttgga ccgcggcgga catggcagct
      541 cagatcacca agcgcaagtg ggaggcggtc catgcggcgg agcagcggag agtctacctg
      601 gagggccggt gcgtggacgg gctccgcaga tacctggaga acgggaagga gacgctgcag
      661 cgcacggacc cccccaagac acatatgacc caccacccca tctctgacca tgaggccacc
      721 ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat
      781 ggggaggacc agacccagga cacggagctc gtggagacca ggcctgcagg ggatggaacc
      841 ttccagaagt gggcggctgt ggtggtgcct tctggagagg agcagagata cacctgccat
      901 gtgcagcatg agggtctgcc caagcccctc accctgagat gggagctgtc ttcccagccc
      961 ggatccctgc atcatattct ggatgcacag aaaatggtgt ggaatcatcg ttaaaagctt
     1021 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     1081 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     1141 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     1201 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1261 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1321 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1381 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1441 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1501 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1561 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1621 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc aggtggcact tttcggggaa
     1681 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     1741 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     1801 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     1861 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     1921 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     1981 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     2041 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     2101 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     2161 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     2221 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     2281 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     2341 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     2401 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     2461 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     2521 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     2581 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     2641 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2701 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     2761 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     2821 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     2881 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     2941 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3001 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3061 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3121 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3181 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3241 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3301 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3361 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     3421 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     3481 ccttacgcat ctgtgcggta tttcacaccg catatatggt gcactctcag tacaatctgc
     3541 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     3601 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3661 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3721 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     3781 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     3841 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     3901 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     3961 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     4021 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     4081 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     4141 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     4201 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     4261 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     4321 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     4381 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     4441 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     4501 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     4561 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     4621 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     4681 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4741 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     4801 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     4861 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     4921 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     4981 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     5041 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     5101 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     5161 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     5221 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     5281 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     5341 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     5401 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     5461 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     5521 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     5581 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     5641 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     5701 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     5761 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     5821 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     5881 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     5941 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     6001 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     6061 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     6121 gatgccggcc acgatgcgtc cggcgtagag ga
//
