LOCUS       sequence-355801-        5548 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression of class I MHC light chain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5548)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 5548)
  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            "(unpublished)"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: pTCF158 Human beta-2 microglobulin
FEATURES             Location/Qualifiers
     source          1..5548
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     68..87
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        68..86
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    87..111
                     /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             126..148
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             480..497
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(550..568)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      564..611
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      648..1103
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(735..754)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     945..966
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1195..2010)
                     /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(1195..2010)
                     /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     1918..1937
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2002..2021
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2132..2720
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2621..2640
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2874..2891
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2906..3045
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2971..2990)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3131..3153
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3147..3338)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4147..5229)
                     /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     5191..5210
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5230..5307)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5513..5532
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
       61 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
      121 ataattttgt ttaactttaa gaaggagata tacatatgat ccagcgcacc ccgaagatcc
      181 aggtgtacag ccgccaccct gccgagaacg gcaagagcaa cttcctgaac tgctacgtga
      241 gcggcttcca cccgagcgac atcgaggtgg acctgctgaa gaacggcgag cgcatcgaga
      301 aggtggagca cagcgacctg agcttcagca aggactggag cttctacctg ctgtactaca
      361 ccgagttcac cccgaccgag aaggacgagt acgcctgccg cgtgaaccac gtgaccctga
      421 gccagccgaa gatcgtgaag tgggatcgcg acatgtaaaa gcttgcggcc gcactcgagc
      481 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
      541 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
      601 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
      661 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
      721 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
      781 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
      841 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
      901 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
      961 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     1021 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     1081 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     1141 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     1201 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     1261 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     1321 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     1381 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     1441 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     1501 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     1561 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     1621 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     1681 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     1741 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     1801 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     1861 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     1921 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     1981 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     2041 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2101 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2161 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2221 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     2281 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2341 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     2401 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     2461 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     2521 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     2581 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     2641 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     2701 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     2761 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     2821 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     2881 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     2941 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3001 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     3061 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3121 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3181 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     3241 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     3301 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     3361 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     3421 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     3481 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     3541 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     3601 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     3661 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     3721 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     3781 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     3841 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     3901 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     3961 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     4021 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     4081 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     4141 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4201 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     4261 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     4321 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     4381 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     4441 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     4501 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     4561 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     4621 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     4681 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     4741 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     4801 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     4861 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     4921 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     4981 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     5041 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     5101 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     5161 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     5221 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     5281 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     5341 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     5401 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     5461 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     5521 cggcgatata ggcgccagca accgcacc
//
