LOCUS       sequence-357373-        6143 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 6143)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 6143)
  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            "(unpublished)"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: pTCF120 H-2D(b)
FEATURES             Location/Qualifiers
     source          1..6143
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             925..942
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(995..1013)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1009..1056
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1093..1548
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1180..1199)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1390..1411
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1641..2456)
                     /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(1641..2456)
                     /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     2364..2383
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2448..2467
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2578..3166
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3067..3086
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3320..3337
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3352..3494
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3420..3439)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3580..3602
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3596..3787)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4596..5678)
                     /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     5640..5659
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5679..5756)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5962..5981
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6065..6084
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6065..6083
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6084..6108
                     /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             join(6123..6143,1..2)
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gatatacata tgggtccgca tagcatgcgt tattttgaaa ccgcagttag ccgtccgggt
       61 ctggaagaac cgcgttatat tagcgttggt tatgtggata acaaagaatt cgtgcgtttt
      121 gattccgatg cagaaaatcc gcgttatgaa ccgcgtgcac cgtggatgga acaagaaggt
      181 ccggaatatt gggaacgtga aacccagaaa gcaaaaggtc aagaacagtg gtttcgtgtt
      241 agcctgcgta atctgctggg ctattataac cagagtgccg gtggtagcca taccctgcag
      301 cagatgagcg gttgtgatct gggtagcgat tggcgtctgc tgcgtggtta tctgcagttt
      361 gcctatgaag gtcgtgatta tattgccctg aatgaggatc tgaaaacctg gaccgcagca
      421 gatatggcag cacagattac ccgtcgtaaa tgggaacaga gcggtgcagc agaacactat
      481 aaagcatatc tggaaggtga atgtgttgaa tggctgcatc gttatctgaa aaatggtaat
      541 gcaaccctgc tgcgtaccga tagcccgaaa gcacatgtta cccatcatcc gcgtagcaaa
      601 ggtgaagtta ccctgcgttg ttgggcactg ggtttttatc cggcagatat taccctgacc
      661 tggcagctga atggtgaaga actgacccag gatatggaac tggttgaaac ccgtcctgcc
      721 ggtgatggca cctttcagaa atgggcaagc gttgttgttc cgctgggtaa agaacagaat
      781 tatacctgtc gtgtgtatca tgaaggtctg ccggaaccgc tgaccctgcg ctgggaaccg
      841 cctccgagtg gatccctgca tcatattctg gatgcacaga aaatggtgtg gaatcatcgt
      901 taaaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac
      961 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     1021 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     1081 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1141 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1201 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1261 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1321 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1381 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1441 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1501 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttca ggtggcactt
     1561 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1621 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     1681 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     1741 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     1801 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     1861 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     1921 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     1981 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     2041 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     2101 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     2161 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     2221 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     2281 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     2341 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     2401 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     2461 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     2521 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     2581 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     2641 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     2701 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     2761 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     2821 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     2881 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     2941 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3001 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3061 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3121 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3181 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3241 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     3301 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     3361 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt
     3421 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     3481 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3541 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3601 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     3661 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     3721 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     3781 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     3841 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     3901 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     3961 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     4021 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4081 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4141 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4201 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     4261 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     4321 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     4381 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     4441 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     4501 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     4561 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4621 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4681 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     4741 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     4801 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     4861 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     4921 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     4981 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     5041 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     5101 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     5161 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     5221 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     5281 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     5341 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     5401 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     5461 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     5521 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     5581 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     5641 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     5701 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     5761 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     5821 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     5881 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     5941 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     6001 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     6061 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     6121 attttgttta actttaagaa gga
//
