LOCUS       sequence-353141-        7950 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in bacterial cells. Entire CDS,  M1-D1087..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7950)
  AUTHORS   Gileadi, et al
  TITLE     TREAT-AD plasmids 2021
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 7950)
  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            "unpublished"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: EPB41L3A-c000
FEATURES             Location/Qualifiers
     source          1..7950
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             12..34
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             45..62
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             87..107
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2768..2785
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2838..2856)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2852..2899
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2936..3391
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3023..3042)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3233..3254
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3483..4298)
                     /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(3483..4298)
                     /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     4206..4225
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4290..4309
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4420..5008
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4909..4928
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5162..5179
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5194..5333
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5259..5278)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5419..5441
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5435..5626)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6435..7517)
                     /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     7479..7498
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7518..7595)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7801..7820
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7904..7923
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7904..7922
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7923..7947
                     /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)."
ORIGIN
        1 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgcaccat catcatcatc
       61 attcttctgg tgtagatctg ggtaccgaga acctgtactt ccaatccatg acgaccgaat
      121 ctggatcaga ctcggaatcc aagccggacc aggaggccga gccccaggag gcggcggggg
      181 cgcaggggcg cgcgggggcg cccgtgccgg agccgcccaa ggaggagcag cagcaggccc
      241 tggagcagtt cgccgccgct gcagcgcaca gcaccccggt gcggagggag gtcactgaca
      301 aggaacagga gtttgctgcc agggctgcaa aacagctcga atatcagcaa ttagaagacg
      361 ataaactttc tcagaaatca tctagcagta aactctctcg gtctccatta aagattgtca
      421 aaaagcctaa aagcatgcag tgcaaagtga tacttctcga tggatcagaa tatacctgtg
      481 atgtagagaa acgctccaga ggacaagtgc tgtttgataa agtgtgtgaa cacttgaact
      541 tgctagagaa agactacttt gggcttacgt atcgagatgc tgaaaaccag aagaattggt
      601 tggaccctgc taaggaaata aaaaaacagg ttcgaagtgg tgcttggcac ttttcattta
      661 atgtgaaatt ttatccacca gaccctgccc aactatctga agatatcacc aggtactacc
      721 tctgcttgca gttgcgagat gacatcgtgt ccggaaggct gccctgctcc tttgttaccc
      781 tggccttgct gggctcctac actgtccagt cagagctcgg agactatgac ccagatgaat
      841 gtgggagcga ttacattagt gagttccgct ttgcaccaaa ccacactaaa gaactggaag
      901 acaaagtgat cgagctgcac aagagccaca gaggaatgac gccagcagaa gcagagatgc
      961 atttcttgga aaatgccaaa aaattatcaa tgtatggggt agatttacat catgctaagg
     1021 actcagaagg ggtagaaatt atgttaggag tttgtgcaag tggtctgttg atatatcgcg
     1081 accggctgcg aataaacaga tttgcctggc ccaaggttct aaagatttca tacaaacgga
     1141 acaactttta cattaagatc cggccgggag agtttgaaca atttgaaagc accattgggt
     1201 ttaagctgcc aaaccatcga gctgccaagc gtttatggaa agtatgtgtt gagcatcata
     1261 catttttcag actactgtta ccagaagcac ctcccaagaa attcctaacc ttgggttcca
     1321 agtttcgtta tagtggcagg acacaagcgc aaacgagaag agccagtgcg ttgatagatc
     1381 gcccagcacc ttactttgaa cgctcatcca gcaaacgtta taccatgtct cgcagcttgg
     1441 atggagcatc agtgaatgaa aaccatgaaa tatacatgaa ggattctatg tctgctgcag
     1501 aggttggtac tggccagtac gccacaacaa aaggcatctc tcagaccaac ttgatcacca
     1561 ctgtgactcc ggagaagaag gctgaggagg agcgggacga ggaagaggac aaacggagga
     1621 agggggaaga agtcacgccc atctcggcca tccggcacga gggaaagact gacagtgagc
     1681 gcacggacac cgcagccgac ggggagacca ccgccactga ggagctagaa aaaactcaag
     1741 atgacctgat gaaacatcaa accaacatta gcgagctgaa aagaaccttc ttagaaacct
     1801 caacagacac tgccgtaacg aatgaatggg agaagaggct ttccacctcc cccgtgcgac
     1861 tggccgccag gcaggaggat gcccccatga tcgaaccact tgtccctgaa gagaaaatgg
     1921 aaaccaagac ggagtccagt ggaatagaga cggaacccac cgtgcaccac ctgccgctta
     1981 gcactgagaa ggtggtgcag gagaccgtgt tggtggagga gcggcgtgtg gtgcacgcga
     2041 gtggggatgc ttcttactcg gcgggagaca gcggggatgc tgcagcacag cccgcattca
     2101 caggcattaa agggaaagag ggctctgcct tgacggaggg ggctaaagag gaaggagggg
     2161 aggaggtcgc taaagctgtc ctggaacagg aagagacagc cgctgcttcc cgtgagcgac
     2221 aagaggagca gagtgcagcc atccacattt cagaaacttt ggaacaaaaa cctcattttg
     2281 agtcctcaac ggtgaagacg gaaaccatca gttttggcag tgtttcaccg ggaggagtaa
     2341 agctagaaat ttccacgaag gaagtgccag tagttcacac cgaaaccaaa accatcacat
     2401 atgaatcatc acaggtcgat ccaggcacag atctggagcc aggcgtgctg atgagtgcac
     2461 agacgatcac atctgaaacc accagtacca ccaccactac gcacatcacc aaaactgtga
     2521 aagggggcat ttcagagaca agaattgaga agcgaatagt catcacgggg gatgcagaca
     2581 ttgaccatga ccaggcgctg gctcaggcaa ttaaagaggc caaagagcag caccctgaca
     2641 tgtcagtgac caaagtagtg gtccataaag agacagagat cacaccagaa gatggagagg
     2701 attgacagta aaggtggata cggatccgaa ttcgagctcc gtcgacaagc ttgcggccgc
     2761 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
     2821 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     2881 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     2941 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3001 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3061 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3121 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3181 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3241 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3301 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3361 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     3421 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     3481 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     3541 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     3601 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     3661 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     3721 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     3781 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     3841 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     3901 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     3961 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     4021 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     4081 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     4141 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     4201 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     4261 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     4321 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     4381 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4441 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4501 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4561 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4621 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4681 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4741 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4801 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4861 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4921 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4981 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5041 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5101 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     5161 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     5221 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     5281 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     5341 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5401 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5461 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     5521 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     5581 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     5641 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     5701 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     5761 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     5821 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     5881 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     5941 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     6001 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     6061 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     6121 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     6181 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     6241 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     6301 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     6361 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     6421 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     6481 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     6541 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     6601 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     6661 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     6721 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     6781 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     6841 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     6901 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     6961 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     7021 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     7081 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     7141 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     7201 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     7261 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     7321 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     7381 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     7441 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     7501 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     7561 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     7621 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     7681 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     7741 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     7801 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     7861 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     7921 ggggaattgt gagcggataa caattcccct
//
