LOCUS       sequence-409097-        6139 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 6139)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 6139)
  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            "(unpublished)"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: pTCF79 H-2D(b) (CD8 mut)
FEATURES             Location/Qualifiers
     source          1..6139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     589..608
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(628..705)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     911..930
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1014..1033
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1014..1032
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1033..1057
                     /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             1072..1094
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2017..2034
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2087..2105)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2101..2148
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2185..2640
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2272..2291)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2482..2503
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2732..3547)
                     /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(2732..3547)
                     /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     3455..3474
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3539..3558
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3669..4257
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4158..4177
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4411..4428
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4443..4582
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4508..4527)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4668..4690
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4684..4875)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5684..6139,1..627))
                     /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 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
       61 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
      121 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
      181 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
      241 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
      301 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
      361 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
      421 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
      481 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
      541 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
      601 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
      661 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
      721 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
      781 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
      841 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
      901 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
      961 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     1021 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     1081 ctttaagaag gagatataca tatgggccca cactcgatgc ggtatttcga gaccgccgtg
     1141 tcccggcccg gcctcgagga gccccggtac atctctgtcg gctatgtgga caacaaggag
     1201 ttcgtgcgct tcgacagcga cgcggagaat ccgagatatg agccgcgggc gccgtggatg
     1261 gagcaggagg ggccggagta ttgggagcgg gaaacacaga aagccaaggg ccaagagcag
     1321 tggttccgag tgagcctgag gaacctgctc ggctactaca accagagcgc gggcggctct
     1381 cacacactcc agcagatgtc tggctgtgac ttggggtcgg actggcgcct cctccgcggg
     1441 tacctgcagt tcgcctatga aggccgcgat tacatcgccc tgaacgaaga cctgaaaacg
     1501 tggacggcgg cggacatggc ggcgcagatc acccgacgca agtgggagca gagtggtgct
     1561 gcagagcatt acaaggccta cctggagggc gagtgcgtgg agtggctcca cagatacctg
     1621 aagaacggga acgcgacgct gctgcgcaca gattccccaa aggcacatgt gacccatcac
     1681 cccagatcta aaggtgaagt caccctgagg tgctgggccc tgggcttcta ccctgctgac
     1741 atcaccctga cctggcagtt gaatggggag gagctgaccc agaaaatgga gcttgtggag
     1801 accaggcctg caggggatgg aaccttccag aagtgggcat ctgtggtggt gcctcttggg
     1861 aaggagcaga attacacatg ccgtgtgtac catgaggggc tgcctgagcc cctcaccctg
     1921 agatgggagc ctcctccgtc cggatccctg catcatattc tggatgcaca gaaaatggtg
     1981 tggaatcatc gttaaaagct tgcggccgca ctcgagcacc accaccacca ccactgagat
     2041 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     2101 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     2161 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2221 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2281 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2341 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2401 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2461 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2521 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2581 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2641 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2701 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     2761 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     2821 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     2881 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     2941 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     3001 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     3061 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     3121 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     3181 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     3241 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     3301 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     3361 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3421 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     3481 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     3541 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     3601 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3661 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3721 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3781 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3841 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3901 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3961 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4021 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4081 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4141 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4201 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4261 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4321 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4381 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4441 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     4501 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4561 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4621 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4681 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4741 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     4801 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     4861 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     4921 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     4981 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     5041 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     5101 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     5161 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     5221 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     5281 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     5341 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     5401 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     5461 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     5521 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     5581 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     5641 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     5701 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5761 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     5821 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     5881 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     5941 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     6001 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     6061 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     6121 gttccgctat cggctgaat
//
