LOCUS       sequence-355847-        6139 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 6139)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 6139)
  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            "(unpublished)"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: pTCF170 H-2L(d)
FEATURES             Location/Qualifiers
     source          1..6139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     187..206
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     290..309
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        290..308
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    309..333
                     /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             348..370
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1293..1310
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1363..1381)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1377..1424
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1461..1916
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1548..1567)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1758..1779
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2008..2823)
                     /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(2008..2823)
                     /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     2731..2750
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2815..2834
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2945..3533
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3434..3453
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3687..3704
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3719..3858
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3784..3803)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3944..3966
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3960..4151)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4960..6042)
                     /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     6004..6023
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6043..6120)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
       61 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
      121 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
      181 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
      241 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
      301 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
      361 aagaaggaga tatacatatg ggtccgcata gcatgcgtta ttttgaaacc gcagttagcc
      421 gtccgggtct gggtgaaccg cgttatatta gcgttggtta tgtggataac aaagaattcg
      481 tgcgttttga ttccgatgca gaaaatccgc gttatgaacc gcaggcaccg tggatggaac
      541 aagaaggtcc ggaatattgg gaacgtatta cccagattgc aaaaggtcaa gaacagtggt
      601 ttcgtgttaa tctgcgtacc ctgctgggct attataacca gagtgccggt ggcacccata
      661 ccctgcagtg gatgtatggt tgtgatgttg gtagtgatgg tcgtctgctg cgtggttatg
      721 aacagtttgc ctatgatggt tgcgattata ttgccctgaa tgaggatctg aaaacctgga
      781 ccgcagcaga tatggcagca cagattaccc gtcgtaaatg ggaacaggca ggcgcagcag
      841 aatactatcg tgcatatctg gaaggtgaat gtgttgaatg gctgcatcgt tatctgaaaa
      901 atggtaatgc aaccctgctg cgtaccgata gcccgaaagc acatgttacc catcatccgc
      961 gtagcaaagg tgaagttacc ctgcgttgtt gggcactggg tttttatccg gcagatatta
     1021 ccctgacctg gcagctgaat ggtgaagaac tgacccagga tatggaactg gttgaaaccc
     1081 gtcctgccgg tgatggcacc tttcagaaat gggcaagcgt tgttgttccg ctgggtaaag
     1141 aacagaatta tacctgtcgt gtgtatcatg aaggtctgcc ggaaccgctg accctgcgct
     1201 gggaaccgcc tccgagtgga tccctgcatc atattctgga tgcacagaaa atggtgtgga
     1261 atcatcgtta aaagcttgcg gccgcactcg agcaccacca ccaccaccac tgagatccgg
     1321 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     1381 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     1441 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     1501 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     1561 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     1621 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     1681 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     1741 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     1801 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     1861 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     1921 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     1981 aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg
     2041 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga
     2101 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat
     2161 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc
     2221 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat
     2281 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc
     2341 aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt
     2401 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc
     2461 aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg
     2521 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg
     2581 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc
     2641 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg
     2701 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc
     2761 agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct
     2821 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa
     2881 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     2941 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3001 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3061 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     3121 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3181 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3241 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3301 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3361 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3421 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3481 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3541 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3601 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     3661 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     3721 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     3781 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     3841 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     3901 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     3961 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     4021 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     4081 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     4141 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     4201 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     4261 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     4321 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     4381 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     4441 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     4501 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     4561 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     4621 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     4681 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     4741 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     4801 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     4861 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     4921 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4981 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5041 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     5101 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     5161 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     5221 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     5281 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     5341 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     5401 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     5461 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     5521 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     5581 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     5641 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     5701 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     5761 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     5821 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     5881 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     5941 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     6001 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     6061 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     6121 cgggatctcg acgctctcc
//
