LOCUS       sequence-357367-        6146 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 6146)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 6146)
  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            "(unpublished)"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: pTCF236 HLA-B*40:01
FEATURES             Location/Qualifiers
     source          1..6146
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     199..218
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(238..315)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     521..540
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     624..643
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        624..642
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    643..667
                     /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             682..704
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1630..1647
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1700..1718)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1714..1761
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1798..2253
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1885..1904)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2095..2116
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2346..3161)
                     /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(2346..3161)
                     /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     3069..3088
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3153..3172
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3283..3871
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3772..3791
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4025..4042
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4057..4199
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4125..4144)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4285..4307
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4301..4492)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5301..6146,1..237))
                     /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"
ORIGIN
        1 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
       61 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
      121 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
      181 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
      241 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
      301 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
      361 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
      421 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
      481 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
      541 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
      601 tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
      661 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgggctct
      721 cattccatgc gctacttcca tacagctatg tcgcgtcccg gtcgcgggga gccacgcttc
      781 attacggtag gctatgtgga cgatacactt tttgttcgtt tcgacagtga cgcgacctcg
      841 ccgcgtaaag aaccgcgtgc gccttggatc gagcaagaag gcccagagta ctgggaccgt
      901 gagacacaga tttcgaagac aaacacacaa acataccgcg aatccttgcg taatctgcgt
      961 ggctattaca atcagtcaga ggcaggcagt cacacgctgc aacgcatgta tggttgtgat
     1021 gttgggccag acgggcgttt attacgcggg cataaccagt acgcctatga cggtaaagac
     1081 tatattgctc tgaatgagga cttgcgttct tggacggcgg ctgatacggc tgctcaaatt
     1141 agccaacgta aattggaggc ggcgcgcgta gcggaacagc tgcgcgccta cttggagggt
     1201 gagtgtgttg aatggttgcg tcgctacctt gagaacggaa aggacaaatt ggagcgtgcc
     1261 gatccaccta aaactcatgt gacccaccat cctattagcg atcacgaggc gaccttacgt
     1321 tgttgggcat taggctttta tcccgccgag atcaccctta cttggcagcg cgatggggag
     1381 gaccaaactc aggatacgga actggtagaa acgcgtcctg ctggagatcg cacattccag
     1441 aagtgggcag cggttgttgt tccttcggga gaagagcagc gttatacttg ccatgtacaa
     1501 catgaagggt tacctaaacc actgacactt cgttgggaac catcgtcgca gagtggatcc
     1561 ctgcatcata ttctggatgc acagaaaatg gtgtggaatc atcgttaaaa gcttgcggcc
     1621 gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     1681 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1741 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     1801 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     1861 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     1921 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     1981 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2041 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2101 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     2161 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     2221 cgaattttaa caaaatatta acgtttacaa tttcaggtgg cacttttcgg ggaaatgtgc
     2281 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     2341 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     2401 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2461 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2521 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2581 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     2641 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     2701 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     2761 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     2821 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     2881 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     2941 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     3001 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     3061 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     3121 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     3181 tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt
     3241 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3301 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3361 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3421 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3481 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3541 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3601 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3661 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3721 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3781 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3841 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3901 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3961 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4021 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     4081 gcatctgtgc ggtatttcac accgcatata tggtgcactc tcagtacaat ctgctctgat
     4141 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4201 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4261 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4321 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     4381 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     4441 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4501 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     4561 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     4621 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     4681 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4741 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4801 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4861 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4921 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     4981 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     5041 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     5101 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     5161 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     5221 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     5281 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5341 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     5401 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     5461 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     5521 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     5581 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     5641 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5701 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5761 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5821 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5881 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5941 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     6001 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     6061 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     6121 tcggcgcgag atttaatcgc cgcgac
//
