LOCUS       sequence-353174-        6601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in bacterial cells. Entire CDS,  M1-Y329..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6601)
  AUTHORS   Gileadi, et al
  TITLE     TREAT-AD plasmids 2021
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6601)
  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            "unpublished"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: CNN3A-c002
FEATURES             Location/Qualifiers
     source          1..6601
                     /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             1374..1418
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1419..1436
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1489..1507)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1503..1550
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1587..2042
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1674..1693)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1884..1905
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2134..2949)
                     /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(2134..2949)
                     /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     2857..2876
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2941..2960
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3071..3659
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3560..3579
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3813..3830
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3845..3984
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3910..3929)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4070..4092
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4086..4277)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5086..6168)
                     /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     6130..6149
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6169..6246)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6452..6471
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6555..6574
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6555..6573
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6574..6598
                     /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 aggtatgacc cacttcaaca agggcccttc ctatgggctc tcggccgaag
      421 tcaagaacaa gattgcttcc aagtatgatc atcaggcaga agaagatctt cgcaattgga
      481 tagaagaggt gacaggcatg agcattggcc ccaacttcca gctgggctta aaggatggca
      541 tcatcctctg cgaacttata aacaagctac agccaggctc agtgaagaag gtcaacgagt
      601 cctcactgaa ctggcctcag ttggagaata ttggcaactt tattaaagct attcaggctt
      661 atggtatgaa gccacatgac atattcgaag caaatgatct ttttgagaat ggaaacatga
      721 cccaggttca gactactctg gtggctctag caggtctggc taaaacaaaa ggattccata
      781 caaccattga cattggagtt aagtatgcag aaaaacaaac aagacgtttt gatgaaggaa
      841 aattaaaagc tggccaaagt gtaattggtc tgcagatggg aaccaacaaa tgtgccagcc
      901 aggcaggtat gacagcttac gggactagga ggcatcttta tgatcccaaa atgcaaactg
      961 acaaaccttt tgaccagacc acaattagtc tgcagatggg cactaataaa ggagccagcc
     1021 aggcagggat gttagcacca ggtaccagaa gagacatcta tgatcagaag ctaacattac
     1081 agccggtgga caactcgaca atttccctac agatgggtac caacaaagtt gcttcccaga
     1141 aaggaatgag tgtgtatggg cttgggcggc aagtatatga tcccaaatac tgtgctgctc
     1201 ctacagaacc tgtcattcac aacggaagcc aaggaacagg aacaaatggt tcggaaatca
     1261 gtgatagtga ttatcaggca gaataccctg atgagtatca tggcgagtac caggatgact
     1321 accccagaga ttaccaatat agcgaccaag gcattgatta ttgaagcggt ggcggtctga
     1381 acgacatctt cgaggctcag aaaatcgaat ggcacgaaca ccaccaccac caccactgag
     1441 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     1501 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     1561 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     1621 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     1681 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     1741 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     1801 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     1861 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     1921 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     1981 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     2041 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2101 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
     2161 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     2221 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     2281 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     2341 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     2401 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     2461 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     2521 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     2581 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     2641 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     2701 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     2761 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     2821 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     2881 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     2941 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     3001 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3061 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3121 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3181 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     3241 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3301 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     3361 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3421 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3481 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3541 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3601 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3661 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3721 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3781 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     3841 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     3901 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     3961 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     4021 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4081 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     4141 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     4201 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     4261 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     4321 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     4381 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     4441 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     4501 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     4561 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     4621 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     4681 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     4741 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     4801 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     4861 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     4921 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     4981 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     5041 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     5101 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5161 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     5221 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     5281 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     5341 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     5401 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     5461 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     5521 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     5581 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     5641 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     5701 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     5761 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     5821 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     5881 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     5941 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     6001 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     6061 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     6121 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     6181 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     6241 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     6301 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     6361 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     6421 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     6481 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     6541 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     6601 t
//
