LOCUS       sequence-357359-        6152 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 6152)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 6152)
  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            "(unpublished)"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: pTCF3 H-2D(d)
FEATURES             Location/Qualifiers
     source          1..6152
                     /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             1042..1059
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1112..1130)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1126..1173
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1210..1665
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1297..1316)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1507..1528
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1758..2573)
                     /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(1758..2573)
                     /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     2481..2500
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2565..2584
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2695..3283
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3184..3203
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3437..3454
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3469..3611
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3537..3556)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3697..3719
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3713..3904)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4713..5795)
                     /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     5757..5776
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5796..5873)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6079..6098
                     /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 ctcttagata tttcgtcacc gccgtgtccc ggcccggctt cggggagccc
      181 cggtacatgg aagtcggcta cgtggacaac acggagttcg tgcgcttcga cagcgacgcg
      241 gagaatccga gatatgagcc gcgggcgcgg tggatagagc aggaggggcc ggagtattgg
      301 gagcgggaga cacggagagc caagggcaat gagcagagtt tccgagtgga cctgaggacc
      361 gcgctgcgct actacaacca gagcgcgggc ggctctcaca cactccagtg gatggctggc
      421 tgtgacgtgg agtcggacgg gcgcctcctc cgcgggtact ggcagttcgc ctacgacggc
      481 tgcgattaca tcgccctgaa cgaagacctg aaaacgtgga cggcggcgga catggcggcg
      541 cagatcaccc gacgcaagtg ggagcaggct ggtgctgcag agagagaccg ggcctacctg
      601 gagggcgagt gcgtggagtg gctccgcaga tacctgaaga acgggaatgc tacgctgctg
      661 cgcacagatc ccccaaaggc ccatgtgacc catcaccgca gacctgaagg tgatgtcacc
      721 ctgaggtgct gggccctggg cttctaccct gctgacatca ccctgacctg gcagttgaat
      781 ggggaggagc tgacccagga aatggagctt gtggagacca ggcctgcagg ggatggaacc
      841 ttccagaagt gggcatctgt ggtggtgcct cttgggaagg agcagaagta cacatgccat
      901 gtggaacatg aggggctgcc tgagcccctc accctgagat ggggcaagga ggagcctcct
      961 tcatccggat ccctgcatca tattctggat gcacagaaaa tggtgtggaa tcatcgttaa
     1021 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     1081 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     1141 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     1201 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1261 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     1321 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     1381 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     1441 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     1501 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     1561 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     1621 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttcaggt ggcacttttc
     1681 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     1741 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
     1801 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     1861 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     1921 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     1981 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     2041 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     2101 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     2161 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     2221 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     2281 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     2341 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     2401 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     2461 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     2521 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     2581 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg
     2641 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2701 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2761 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2821 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     2881 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2941 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3001 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3061 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3121 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3181 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3241 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3301 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3361 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3421 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     3481 ttttctcctt acgcatctgt gcggtatttc acaccgcata tatggtgcac tctcagtaca
     3541 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     3601 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3661 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     3721 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     3781 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     3841 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     3901 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     3961 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     4021 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     4081 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     4141 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     4201 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     4261 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     4321 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     4381 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     4441 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     4501 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     4561 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     4621 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     4681 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4741 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4801 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     4861 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     4921 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     4981 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     5041 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     5101 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     5161 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     5221 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     5281 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     5341 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     5401 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     5461 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     5521 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     5581 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     5641 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     5701 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     5761 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     5821 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     5881 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     5941 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     6001 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     6061 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     6121 gccggtgatg ccggccacga tgcgtccggc gt
//
