LOCUS       sequence-357369-        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  JOURNAL   (unpublished)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            "(unpublished)"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: pTCF7 HLA-B*07:02
FEATURES             Location/Qualifiers
     source          1..6146
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    40..64
                     /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             79..101
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1027..1044
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1097..1115)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1111..1158
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1195..1650
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1282..1301)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1492..1513
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1743..2558)
                     /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(1743..2558)
                     /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     2466..2485
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2550..2569
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2680..3268
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3169..3188
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3422..3439
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3454..3596
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3522..3541)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3682..3704
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3698..3889)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4698..5780)
                     /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     5742..5761
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5781..5858)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6064..6083
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
       61 ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat gggttctcat
      121 tctatgagat atttctacac ctccgtgtcc cggcccggcc gcggggagcc ccgcttcatc
      181 tcagtgggct acgtggacga cacccagttc gtgaggttcg acagcgacgc cgcgagtccg
      241 agagaggagc cgcgggcgcc gtggatagag caggaggggc cggagtattg ggaccggaac
      301 acacagatct acaaggccca ggcacagact gaccgagaga gcctgcggaa cctgcgcggc
      361 tactacaacc agagcgaggc cgggtctcac accctccaga gcatgtacgg ctgcgacgtg
      421 gggccggacg ggcgcctcct ccgcgggcat gaccagtacg cctacgacgg caaggattac
      481 atcgccctga acgaggacct gcgctcctgg accgccgcgg acacggcggc tcagatcacc
      541 cagcgcaagt gggaggcggc ccgtgaggcg gagcagcgga gagcctacct ggagggcgag
      601 tgcgtggagt ggctccgcag atacctggag aacgggaagg acaagctgga gcgcgctgac
      661 cccccaaaga cacacgtgac ccaccacccc atctctgacc atgaggccac cctgaggtgc
      721 tgggccctgg gtttctaccc tgcggagatc acactgacct ggcagcggga tggcgaggac
      781 caaactcagg acactgagct tgtggagacc agaccagcag gagatagaac cttccagaag
      841 tgggcagctg tggtggtgcc ttctggagaa gagcagagat acacatgcca tgtacagcat
      901 gaggggctgc cgaagcccct caccctgaga tgggagccgt cttcccagtc cggatccctg
      961 catcatattc tggatgcaca gaaaatggtg tggaatcatc gttaaaagct tgcggccgca
     1021 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     1081 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     1141 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     1201 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1261 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     1321 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     1381 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1441 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1501 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     1561 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     1621 attttaacaa aatattaacg tttacaattt caggtggcac ttttcgggga aatgtgcgcg
     1681 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     1741 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     1801 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     1861 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     1921 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     1981 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     2041 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     2101 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     2161 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     2221 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     2281 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     2341 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     2401 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     2461 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     2521 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     2581 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     2641 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     2701 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2761 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2821 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2881 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2941 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3001 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3061 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3121 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3181 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3241 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3301 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3361 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3421 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     3481 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc
     3541 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     3601 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3661 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3721 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     3781 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     3841 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     3901 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     3961 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     4021 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     4081 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     4141 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     4201 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     4261 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     4321 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     4381 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     4441 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     4501 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     4561 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     4621 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     4681 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4741 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     4801 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     4861 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     4921 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     4981 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     5041 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     5101 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     5161 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     5221 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     5281 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     5341 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     5401 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     5461 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     5521 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     5581 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     5641 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     5701 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     5761 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     5821 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     5881 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     5941 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     6001 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     6061 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     6121 cacgatgcgt ccggcgtaga ggatcg
//
