LOCUS       sequence-355083-        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  JOURNAL   (unpublished)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            "(unpublished)"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: pTCF200 Mouse beta-2 microglobulin
FEATURES             Location/Qualifiers
     source          1..5548
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(418..1500)
                     /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     1462..1481
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1501..1578)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1784..1803
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1887..1906
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1887..1905
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1906..1930
                     /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             1945..1967
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2299..2316
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2369..2387)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2383..2430
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2467..2922
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2554..2573)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2764..2785
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3014..3829)
                     /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(3014..3829)
                     /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     3737..3756
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3821..3840
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3951..4539
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4440..4459
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4693..4710
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4725..4864
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4790..4809)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4950..4972
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4966..5157)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
       61 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
      121 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
      181 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
      241 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
      301 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
      361 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
      421 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
      481 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
      541 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
      601 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
      661 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
      721 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
      781 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
      841 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
      901 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
      961 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     1021 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     1081 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     1141 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     1201 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     1261 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     1321 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     1381 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     1441 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     1501 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     1561 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     1621 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     1681 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     1741 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     1801 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     1861 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     1921 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatatgatc
     1981 cagaaaaccc ctcagattca ggtttattcg cgtcacccgc cggagaatgg caaaccaaac
     2041 atcttgaact gctatgtcac gcagttccac ccgccgcata ttgagattca aatgttaaag
     2101 aatggcaaaa agatcccaaa agtcgaaatg tcagatatga gctttagcaa ggattggtca
     2161 ttttatatcc tcgcccatac tgaatttaca ccaacggaga ccgataccta tgcctgccgt
     2221 gtgaaacacg cgtctatggc ggaaccgaag accgtttatt gggatcgtga catgtaaaag
     2281 cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     2341 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     2401 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     2461 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2521 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2581 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2641 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2701 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2761 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2821 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     2881 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     2941 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3001 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     3061 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     3121 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     3181 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     3241 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     3301 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     3361 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     3421 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     3481 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     3541 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     3601 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     3661 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     3721 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     3781 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     3841 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     3901 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3961 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4021 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4081 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     4141 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4201 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4261 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4321 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4381 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4441 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4501 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4561 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4621 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4681 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     4741 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     4801 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     4861 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4921 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4981 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     5041 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     5101 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     5161 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     5221 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     5281 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     5341 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     5401 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     5461 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     5521 tcacgttcgc tcgcgtatcg gtgattca
//
