LOCUS       sequence-353209-        6321 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in bacterial cells. FERM domain,  M1-Q322..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6321)
  AUTHORS   Gileadi, et al
  TITLE     TREAT-AD plasmids 2021
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6321)
  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            "unpublished"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: EZRA-c004
FEATURES             Location/Qualifiers
     source          1..6321
                     /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             1139..1156
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1209..1227)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1223..1270
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1307..1762
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1394..1413)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1604..1625
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1854..2669)
                     /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(1854..2669)
                     /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     2577..2596
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2661..2680
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2791..3379
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3280..3299
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3533..3550
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3565..3704
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3630..3649)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3790..3812
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3806..3997)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4806..5888)
                     /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     5850..5869
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5889..5966)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6172..6191
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6275..6294
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6275..6293
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6294..6318
                     /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 ccgaaaccaa
      121 tcaatgtccg agttaccacc atggatgcag agctggagtt tgcaatccag ccaaatacaa
      181 ctggaaaaca gctttttgat caggtggtaa agactatcgg cctccgggaa gtgtggtact
      241 ttggcctcca ctatgtggat aataaaggat ttcctacctg gctgaagctg gataagaagg
      301 tgtctgccca ggaggtcagg aaggagaatc ccctccagtt caagttccgg gccaagttct
      361 accctgaaga tgtggctgag gagctcatcc aggacatcac ccagaaactt ttcttcctcc
      421 aagtgaagga aggaatcctt agcgatgaga tctactgccc ccctgagact gccgtgctct
      481 tggggtccta cgctgtgcag gccaagtttg gggactacaa caaagaagtg cacaagtctg
      541 ggtacctcag ctctgagcgg ctgatccctc aaagagtgat ggaccagcac aaacttacca
      601 gggaccagtg ggaggaccgg atccaggtgt ggcatgcgga acaccgtggg atgctcaaag
      661 ataatgctat gttggaatac ctgaagattg ctcaggacct ggaaatgtat ggaatcaact
      721 atttcgagat aaaaaacaag aaaggaacag acctttggct tggagttgat gcccttggac
      781 tgaatattta tgagaaagat gataagttaa ccccaaagat tggctttcct tggagtgaaa
      841 tcaggaacat ctctttcaat gacaaaaagt ttgtcattaa acccatcgac aagaaggcac
      901 ctgactttgt gttttatgcc ccacgtctga gaatcaacaa gcggatcctg cagctctgca
      961 tgggcaacca tgagttgtat atgcgccgca ggaagcctga caccatcgag gtgcagcaga
     1021 tgaaggccca ggcccgggag gagaagcatc agaagcagct ggagcggcaa cagtgacagt
     1081 aaaggtggat acggatccga attcgagctc cgtcgacaag cttgcggccg cactcgagca
     1141 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     1201 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     1261 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     1321 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1381 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     1441 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     1501 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     1561 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     1621 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     1681 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     1741 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1801 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     1861 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     1921 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     1981 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     2041 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     2101 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     2161 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     2221 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     2281 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     2341 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     2401 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     2461 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     2521 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     2581 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     2641 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     2701 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     2761 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     2821 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     2881 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     2941 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3001 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3061 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3121 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3181 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3241 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3301 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3361 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3421 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3481 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     3541 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3601 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3661 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     3721 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3781 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3841 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     3901 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     3961 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     4021 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     4081 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     4141 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     4201 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     4261 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     4321 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     4381 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     4441 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     4501 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     4561 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     4621 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     4681 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     4741 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     4801 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4861 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     4921 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     4981 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     5041 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     5101 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     5161 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     5221 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     5281 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     5341 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     5401 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     5461 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     5521 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     5581 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     5641 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     5701 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     5761 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     5821 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     5881 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     5941 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     6001 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     6061 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     6121 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     6181 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     6241 ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg
     6301 tgagcggata acaattcccc t
//
