LOCUS       sequence-357371-        5552 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 5552)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 5552)
  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            "(unpublished)"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..5552
                     /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(1196..2011)
                     /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(1196..2011)
                     /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     1919..1938
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2003..2022
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2133..2721
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2622..2641
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2875..2892
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2907..3049
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2975..2994)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3135..3157
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3151..3342)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4151..5233)
                     /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     5195..5214
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5234..5311)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5517..5536
                     /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 acgtttacaa tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1141 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     1201 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     1261 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     1321 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     1381 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     1441 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     1501 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     1561 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     1621 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     1681 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     1741 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     1801 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     1861 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     1921 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     1981 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     2041 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2101 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2161 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     2221 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     2281 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     2341 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     2401 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     2461 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     2521 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     2581 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     2641 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     2701 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     2761 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     2821 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     2881 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     2941 ggtatttcac accgcatata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3001 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     3061 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     3121 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     3181 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     3241 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     3301 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     3361 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     3421 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     3481 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     3541 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     3601 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     3661 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     3721 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     3781 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     3841 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     3901 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     3961 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     4021 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     4081 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     4141 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4201 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     4261 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     4321 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     4381 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     4441 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     4501 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     4561 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     4621 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     4681 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     4741 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     4801 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     4861 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     4921 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     4981 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     5041 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     5101 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     5161 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     5221 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     5281 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     5341 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     5401 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     5461 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     5521 atgtcggcga tataggcgcc agcaaccgca cc
//
