LOCUS       sequence-353138-        6525 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in bacterial cells. FERM domain,  M1-L390..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6525)
  AUTHORS   Gileadi, et al
  TITLE     TREAT-AD plasmids 2021
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6525)
  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            "unpublished"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-c001
FEATURES             Location/Qualifiers
     source          1..6525
                     /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             1343..1360
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1413..1431)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1427..1474
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1511..1966
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1598..1617)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1808..1829
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2058..2873)
                     /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(2058..2873)
                     /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     2781..2800
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2865..2884
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2995..3583
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3484..3503
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3737..3754
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3769..3908
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3834..3853)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3994..4016
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4010..4201)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5010..6092)
                     /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     6054..6073
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6093..6170)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6376..6395
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6479..6498
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6479..6497
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6498..6522
                     /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 actactgtga cagtaaaggt ggatacggat ccgaattcga gctccgtcga
     1321 caagcttgcg gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa
     1381 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
     1441 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt
     1501 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     1561 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     1621 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     1681 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     1741 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     1801 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     1861 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     1921 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     1981 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2041 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
     2101 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     2161 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     2221 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     2281 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     2341 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     2401 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     2461 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     2521 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     2581 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     2641 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     2701 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     2761 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     2821 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     2881 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg
     2941 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3001 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3061 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3121 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     3181 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3241 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3301 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3361 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3421 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3481 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3541 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3601 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3661 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3721 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     3781 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     3841 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     3901 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3961 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4021 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     4081 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     4141 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     4201 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     4261 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     4321 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     4381 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     4441 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     4501 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     4561 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     4621 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     4681 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     4741 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     4801 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     4861 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     4921 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     4981 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5041 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5101 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     5161 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     5221 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     5281 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     5341 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     5401 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     5461 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     5521 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     5581 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     5641 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     5701 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     5761 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     5821 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     5881 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     5941 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     6001 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     6061 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     6121 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     6181 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     6241 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     6301 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     6361 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     6421 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta
     6481 atacgactca ctatagggga attgtgagcg gataacaatt cccct
//
