LOCUS       sequence-353181-        6439 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in bacterial cells. Coiled-coil domains, 
            M1-L275..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6439)
  AUTHORS   Gileadi, et al
  TITLE     TREAT-AD plasmids 2021
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6439)
  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            "unpublished"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: STX4AA-c005
FEATURES             Location/Qualifiers
     source          1..6439
                     /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             54..71
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1212..1256
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1257..1274
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1327..1345)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1341..1388
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1425..1880
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1512..1531)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1722..1743
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1972..2787)
                     /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(1972..2787)
                     /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     2695..2714
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2779..2798
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2909..3497
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3398..3417
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3651..3668
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3683..3822
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3748..3767)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3908..3930
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3924..4115)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4924..6006)
                     /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     5968..5987
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6007..6084)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6290..6309
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6393..6412
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6393..6411
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6412..6436
                     /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 tatgtgcagc agccatcatc
       61 atcatcatca cggcagcggc agcggctctg accaggaggc aaaaccttca actgaggact
      121 tgggggataa gaaggaaggt gaatatatta aactcaaagt cattggacag gatagcagtg
      181 agattcactt caaagtgaaa atgacaacac atctcaagaa actcaaagaa tcatactgtc
      241 aaagacaggg tgttccaatg aattcactca ggtttctctt tgagggtcag agaattgctg
      301 ataatcatac tccaaaagaa ctgggaatgg aggaagaaga tgtgattgaa gtttaccagg
      361 agcaaacggg aggtatgcgg gacaggaccc acgagctgag acagggggat gacagctcgg
      421 acgaagagga caaggagcgg gtcgcgctgg tggtgcaccc gggcacggca cggctgggga
      481 gcccggacga ggagttcttc cacaaggtcc ggacaattcg gcagactatt gtcaaactgg
      541 ggaataaagt ccaggagttg gagaaacagc aggtcaccat cctggccacg ccccttcccg
      601 aggagagcat gaagcaggag ctgcagaacc tgcgcgatga gatcaaacag ctggggaggg
      661 agatccgcct gcagctgaag gccatagagc cccagaagga ggaagctgat gagaactata
      721 actccgtcaa cacaagaatg agaaaaaccc agcatggggt cctgtcccag caattcgtgg
      781 agctcatcaa caagtgcaat tcaatgcagt ccgaataccg ggagaagaac gtggagcgga
      841 ttcggaggca gctgaagatc accaatgctg ggatggtgtc tgatgaggag ttggagcaga
      901 tgctggacag tgggcaaagc gaggtgtttg tgtccaatat cctgaaggac acgcaggtga
      961 ctcgacaggc cttaaatgag atctcggccc ggcacagtga gatccagcag cttgaacgca
     1021 gtattcgtga gctgcacgac atattcactt ttctggctac cgaagtggag atgcaggggg
     1081 agatgatcaa tcggattgag aagaacatcc tgagctcagc ggactacgtg gaacgtgggc
     1141 aggagcacgt caagacggcc ctggagaacc agaagaaggc gaggaagaag aaagtcttgt
     1201 gaagcggtgg cggtctgaac gacatcttcg aggctcagaa aatcgaatgg cacgaacacc
     1261 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1321 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1381 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     1441 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1501 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1561 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     1621 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     1681 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     1741 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     1801 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     1861 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1921 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
     1981 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     2041 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     2101 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     2161 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     2221 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     2281 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     2341 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     2401 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     2461 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     2521 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     2581 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     2641 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     2701 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     2761 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     2821 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2881 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2941 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3001 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     3061 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3121 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3181 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3241 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3301 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3361 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3421 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3481 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3541 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3601 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3661 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3721 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3781 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3841 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3901 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     3961 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     4021 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     4081 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     4141 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     4201 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     4261 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     4321 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     4381 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     4441 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     4501 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     4561 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     4621 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     4681 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     4741 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     4801 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     4861 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     4921 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4981 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     5041 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     5101 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     5161 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     5221 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     5281 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     5341 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     5401 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     5461 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     5521 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     5581 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     5641 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     5701 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     5761 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     5821 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     5881 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     5941 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     6001 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     6061 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     6121 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     6181 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     6241 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     6301 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     6361 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     6421 agcggataac aattcccct
//
