LOCUS       sequence-355082-        6142 bp    DNA     circular SYN 14-APR-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  JOURNAL   (unpublished)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 Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "(unpublished)"
COMMENT     Sequence Label: pTCF236 HLA-B*40:01
FEATURES             Location/Qualifiers
     source          1..6142
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(417..1499)
                     /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     1461..1480
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1500..1577)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1783..1802
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1886..1905
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1886..1904
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1905..1929
                     /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             1944..1966
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2892..2909
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2962..2980)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2976..3023
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3060..3515
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3147..3166)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3357..3378
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3607..4422)
                     /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(3607..4422)
                     /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     4330..4349
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4414..4433
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4544..5132
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5033..5052
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5286..5303
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5318..5457
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5383..5402)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5543..5565
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5559..5750)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
       61 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
      121 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
      181 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
      241 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
      301 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
      361 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
      421 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
      481 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
      541 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
      601 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
      661 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
      721 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
      781 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
      841 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
      901 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
      961 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     1021 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     1081 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     1141 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     1201 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     1261 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     1321 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     1381 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     1441 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     1501 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     1561 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     1621 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     1681 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     1741 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     1801 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     1861 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     1921 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata catatgggct
     1981 ctcattccat gcgctacttc catacagcta tgtcgcgtcc cggtcgcggg gagccacgct
     2041 tcattacggt aggctatgtg gacgatacac tttttgttcg tttcgacagt gacgcgacct
     2101 cgccgcgtaa agaaccgcgt gcgccttgga tcgagcaaga aggcccagag tactgggacc
     2161 gtgagacaca gatttcgaag acaaacacac aaacataccg cgaatccttg cgtaatctgc
     2221 gtggctatta caatcagtca gaggcaggca gtcacacgct gcaacgcatg tatggttgtg
     2281 atgttgggcc agacgggcgt ttattacgcg ggcataacca gtacgcctat gacggtaaag
     2341 actatattgc tctgaatgag gacttgcgtt cttggacggc ggctgatacg gctgctcaaa
     2401 ttagccaacg taaattggag gcggcgcgcg tagcggaaca gctgcgcgcc tacttggagg
     2461 gtgagtgtgt tgaatggttg cgtcgctacc ttgagaacgg aaaggacaaa ttggagcgtg
     2521 ccgatccacc taaaactcat gtgacccacc atcctattag cgatcacgag gcgaccttac
     2581 gttgttgggc attaggcttt tatcccgccg agatcaccct tacttggcag cgcgatgggg
     2641 aggaccaaac tcaggatacg gaactggtag aaacgcgtcc tgctggagat cgcacattcc
     2701 agaagtgggc agcggttgtt gttccttcgg gagaagagca gcgttatact tgccatgtac
     2761 aacatgaagg gttacctaaa ccactgacac ttcgttggga accatcgtcg cagagtggat
     2821 ccctgcatca tattctggat gcacagaaaa tggtgtggaa tcatcgttaa aagcttgcgg
     2881 ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
     2941 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     3001 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     3061 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3121 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3181 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3241 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3301 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3361 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3421 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3481 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     3541 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     3601 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     3661 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     3721 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     3781 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     3841 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     3901 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     3961 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     4021 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     4081 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     4141 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     4201 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     4261 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     4321 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     4381 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     4441 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
     4501 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4561 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4621 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4681 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4741 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4801 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4861 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4921 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4981 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5041 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5101 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5161 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5221 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     5281 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     5341 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     5401 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     5461 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     5521 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     5581 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     5641 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     5701 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     5761 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     5821 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     5881 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     5941 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     6001 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     6061 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     6121 cgctcgcgta tcggtgattc at
//
