LOCUS       sequence-358091-        6142 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 6142)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 6142)
  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            "(unpublished)"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: pTCF37 HLA-B*35:01
FEATURES             Location/Qualifiers
     source          1..6142
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..49
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        30..48
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    49..73
                     /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             88..110
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1036..1053
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1106..1124)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1120..1167
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1204..1659
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1291..1310)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1501..1522
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1751..2566)
                     /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(1751..2566)
                     /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     2474..2493
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2558..2577
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2688..3276
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3177..3196
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3430..3447
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3462..3601
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3527..3546)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3687..3709
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3703..3894)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4703..5785)
                     /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     5747..5766
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5786..5863)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6069..6088
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
       61 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg
      121 ggttcacatt ctatgagata tttctacacc gccatgtccc ggcccggccg cggggagccc
      181 cgcttcatcg cagtgggcta cgtggacgac acccagttcg tgaggttcga cagcgacgcc
      241 gcgagtccga ggacggagcc ccgggcgcca tggatagagc aggaggggcc ggagtattgg
      301 gaccggaaca cacagatctt caagaccaac acacagactt accgagagag cctgcggaac
      361 ctgcgcggct actacaacca gagcgaggcc gggtctcaca tcatccagag gatgtatggc
      421 tgcgacctgg ggcccgacgg gcgcctcctc cgcgggcatg accagtccgc ctacgacggc
      481 aaggattaca tcgccctgaa cgaggacctg agctcctgga ccgcggcgga caccgcggct
      541 cagatcaccc agcgcaagtg ggaggcggcc cgtgtggcgg agcagctgag agcctacctg
      601 gagggcctgt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag
      661 cgcgcggacc ccccaaagac acacgtgacc caccaccccg tctctgacca tgaggccacc
      721 ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat
      781 ggcgaggacc aaactcagga cactgagctt gtggagacca gaccagcagg agatagaacc
      841 ttccagaagt gggcagctgt ggtggtgcct tctggagaag agcagagata cacatgccat
      901 gtacagcatg aggggctgcc gaagcccctc accctgagat gggagccatc ttcccagtcc
      961 ggatccctgc atcatattct ggatgcacag aaaatggtgt ggaatcatcg ttaaaagctt
     1021 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     1081 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     1141 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     1201 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1261 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1321 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1381 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1441 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1501 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1561 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1621 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     1681 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1741 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     1801 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     1861 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     1921 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     1981 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     2041 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     2101 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     2161 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     2221 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     2281 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     2341 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     2401 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     2461 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     2521 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     2581 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     2641 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     2701 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     2761 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     2821 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     2881 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     2941 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3001 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3061 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3121 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3181 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3241 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3301 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3361 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3421 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     3481 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     3541 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     3601 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3661 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3721 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     3781 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     3841 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     3901 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     3961 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     4021 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     4081 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     4141 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     4201 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     4261 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     4321 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     4381 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     4441 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     4501 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     4561 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     4621 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     4681 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4741 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     4801 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     4861 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     4921 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     4981 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     5041 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     5101 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     5161 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     5221 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     5281 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     5341 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     5401 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     5461 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     5521 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     5581 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     5641 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     5701 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     5761 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     5821 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     5881 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     5941 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     6001 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     6061 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     6121 ccggccacga tgcgtccggc gt
//
