LOCUS       sequence-357366-        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  JOURNAL   (unpublished)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            "(unpublished)"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: pTCF122 Mamu-B*008:01
FEATURES             Location/Qualifiers
     source          1..6146
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     68..87
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        68..86
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    87..111
                     /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             126..148
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1074..1091
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1144..1162)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1158..1205
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1242..1697
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1329..1348)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1539..1560
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1790..2605)
                     /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(1790..2605)
                     /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     2513..2532
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2597..2616
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2727..3315
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3216..3235
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3469..3486
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3501..3643
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3569..3588)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3729..3751
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3745..3936)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4745..5827)
                     /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     5789..5808
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5828..5905)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6111..6130
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
       61 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
      121 ataattttgt ttaactttaa gaaggagata tacatatggg tagccatagc atgcgttatt
      181 ttagcaccag cgttagccgt ccgggtcgtg gtgaaccgcg ttttattagc gttggttatg
      241 ttgatgatac ccagtttgtg cgctttgata gtgatgcaga aagtccgcgt gaagaaccgc
      301 gtgcaccgtg gatggaacaa gaaggtccgg aatattggga agaagaaacc cgtcgcgcaa
      361 aaggtcatgc acagaccgat cgtgcagatc tgggcaccct gcgtggctat tataaccaga
      421 gcgaagcagg ttcacatacc ctgcagacca tgtatggttg tgatctgggt ccggatggtc
      481 gtctgctgcg tggttatcat cagtatgcct atgatggcaa agattatttt gccctgaatg
      541 aagatctgcg tagctggacc gcagcagatg ttgcagcaca gattacccag cgtaaatggg
      601 aagcagcacg tgaagcagaa caggttcgtg cctatctgga aggcacctgt gttgaatggc
      661 tgcgtcgtta cctggaaaat ggtaaagaaa ccctgcagcg tgccgatccg cctaaaaccc
      721 atgttaccca tcatccggtt agcgatcatg aagcaaccct gcgttgttgg gcactgggtt
      781 tttatccggc agaaatcacc ctgacctggc agcgtgatgg tgaagatcag acccaggata
      841 ccgaactggt tgaaacacgt ccgggtggtg atggcacctt tcagaaatgg ggtgcagttg
      901 ttgttccgag cggtgaagaa cagcgctata cctgtcatgt tcagcatgaa ggtctgccgg
      961 aaccgctgac cctgcgctgg gaaccgagca gccagagcgg atccctgcat catattctgg
     1021 atgcacagaa aatggtgtgg aatcatcgtt aaaagcttgc ggccgcactc gagcaccacc
     1081 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     1141 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     1201 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     1261 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     1321 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     1381 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     1441 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     1501 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     1561 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     1621 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     1681 attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     1741 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     1801 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     1861 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     1921 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     1981 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     2041 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     2101 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     2161 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     2221 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     2281 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     2341 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     2401 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     2461 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     2521 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     2581 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     2641 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     2701 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2761 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2821 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     2881 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2941 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3001 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3061 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3121 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3181 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3241 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3301 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3361 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3421 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3481 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3541 tcacaccgca tatatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3601 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     3661 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3721 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3781 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     3841 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     3901 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     3961 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     4021 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     4081 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     4141 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     4201 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     4261 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     4321 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     4381 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     4441 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     4501 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     4561 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     4621 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     4681 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     4741 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4801 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     4861 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     4921 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     4981 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     5041 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5101 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5161 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5221 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5281 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5341 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5401 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5461 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5521 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     5581 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     5641 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     5701 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     5761 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     5821 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     5881 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     5941 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     6001 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     6061 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     6121 gcgatatagg cgccagcaac cgcacc
//
