LOCUS       sequence-357356-        5949 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 5949)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 5949)
  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            "(unpublished)"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..5949
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     450..469
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(489..566)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     772..791
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     875..894
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        875..893
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    894..918
                     /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             933..955
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1878..1895
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1948..1966)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1962..2009
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2046..2501
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2133..2152)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2343..2364
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2594..3409)
                     /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(2594..3409)
                     /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     3317..3336
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3401..3420
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3531..4119
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4020..4039
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4273..4290
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4305..4447
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4373..4392)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4533..4555
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4549..4740)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
       61 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
      121 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
      181 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
      241 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
      301 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
      361 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
      421 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
      481 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
      541 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
      601 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
      661 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
      721 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
      781 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
      841 ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg
      901 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
      961 atatgggtcc gcatagcatg cgttattttg aaaccgcagt tagccgtccg ggtctgggtg
     1021 aaccgcgtta tattagcgtt ggttatgtgg ataacaaaga attcgtgcgt tttgattccg
     1081 atgcagaaaa tccgcgttat gaaccgcagg caccgtggat ggaacaagaa ggtccggaat
     1141 attgggaacg tattacccag attgcaaaag gtcaagaaca gtggtttcgt gttaatctgc
     1201 gtaccctgct gggctattat aaccagagtg ccggtggcac ccataccctg cagtggatgt
     1261 atggttgtga tgttggtagt gatggtcgtc tgctgcgtgg ttatgaacag tttgcctatg
     1321 atggttgcga ttatattgcc ctgaatgagg atctgaaaac ctggaccgca gcagatatgg
     1381 cagcacagat tacccgtcgt aaatgggaac aggcaggcgc agcagaatac tatcgtgcat
     1441 atctggaagg tgaatgtgtt gaatggctgc atcgttatct gaaaaatggt aatgcaaccc
     1501 tgctgcgtac cgatagcccg aaagcacatg ttacccatca tccgcgtagc aaaggtgaag
     1561 ttaccctgcg ttgttgggca ctgggttttt atccggcaga tattaccctg acctggcagc
     1621 tgaatggtga agaactgacc caggatatgg aactggttga aacccgtcct gccggtgatg
     1681 gcacctttca gaaatgggca agcgttgttg ttccgctggg taaagaacag aattatacct
     1741 gtcgtgtgta tcatgaaggt ctgccggaac cgctgaccct gcgctgggaa ccgcctccga
     1801 gtggatccct gcatcatatt ctggatgcac agaaaatggt gtggaatcat cgttaaaagc
     1861 ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     1921 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     1981 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     2041 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2101 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2161 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2221 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2281 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2341 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2401 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2461 atttaacgcg aattttaaca aaatattaac gtttacaatt tcaggtggca cttttcgggg
     2521 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2581 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     2641 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     2701 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     2761 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     2821 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     2881 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     2941 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     3001 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     3061 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     3121 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     3181 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     3241 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     3301 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     3361 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     3421 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     3481 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3541 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3601 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3661 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     3721 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3781 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3841 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3901 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3961 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4021 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4081 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4141 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4201 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4261 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     4321 ctccttacgc atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct
     4381 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4441 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4501 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4561 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     4621 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     4681 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     4741 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     4801 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     4861 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     4921 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     4981 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     5041 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     5101 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5161 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     5221 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     5281 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     5341 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5401 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     5461 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     5521 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5581 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5641 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     5701 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     5761 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     5821 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     5881 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     5941 gcatggttt
//
