LOCUS       sequence-353172-        6000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in bacterial cells. Entire CDS, M1-N215. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6000)
  AUTHORS   Gileadi, et al
  TITLE     TREAT-AD plasmids 2021
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6000)
  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            "unpublished"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: RAB5AA-c003
FEATURES             Location/Qualifiers
     source          1..6000
                     /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             818..835
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(888..906)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      902..949
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      986..1441
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1073..1092)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1283..1304
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1533..2348)
                     /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(1533..2348)
                     /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     2256..2275
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2340..2359
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2470..3058
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2959..2978
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3212..3229
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3244..3383
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3309..3328)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3469..3491
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3485..3676)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4485..5567)
                     /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     5529..5548
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5568..5645)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5851..5870
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5954..5973
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5954..5972
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5973..5997
                     /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 gctagtcgag
      121 gcgcaacaag acccaacggg ccaaatactg gaaataaaat atgccagttc aaactagtac
      181 ttctgggaga gtccgctgtt ggcaaatcaa gcctagtgct tcgttttgtg aaaggccaat
      241 ttcatgaatt tcaagagagt accattgggg ctgcttttct aacccaaact gtatgtcttg
      301 atgacactac agtaaagttt gaaatatggg atacagctgg tcaagaacga taccatagcc
      361 tagcaccaat gtactacaga ggagcacaag cagccatagt tgtatatgat atcacaaatg
      421 aggagtcctt tgcaagagca aaaaattggg ttaaagaact tcagaggcaa gcaagtccta
      481 acattgtaat agctttatcg ggaaacaagg ccgacctagc aaataaaaga gcagtagatt
      541 tccaggaagc acagtcctat gcagatgaca atagtttatt attcatggag acatccgcta
      601 aaacatcaat gaatgtaaat gaaatattca tggcaatagc taaaaaattg ccaaagaatg
      661 aaccacaaaa tccaggagca aattctgcca gaggaagagg agtagacctt accgaaccca
      721 cacaaccaac caggaatcag tgttgtagta actgacagta aaggtggata cggatccgaa
      781 ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactgaga
      841 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
      901 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
      961 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     1021 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     1081 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     1141 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     1201 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     1261 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     1321 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     1381 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     1441 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1501 attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga
     1561 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     1621 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     1681 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     1741 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta
     1801 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     1861 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
     1921 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     1981 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
     2041 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     2101 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     2161 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     2221 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     2281 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata
     2341 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac
     2401 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     2461 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     2521 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     2581 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     2641 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     2701 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     2761 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     2821 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     2881 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     2941 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3001 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     3061 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     3121 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     3181 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     3241 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     3301 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     3361 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     3421 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     3481 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     3541 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     3601 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     3661 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     3721 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     3781 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     3841 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     3901 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     3961 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     4021 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     4081 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     4141 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     4201 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     4261 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     4321 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     4381 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     4441 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     4501 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4561 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     4621 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     4681 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     4741 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     4801 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     4861 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     4921 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     4981 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     5041 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     5101 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     5161 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     5221 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     5281 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     5341 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     5401 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     5461 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     5521 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     5581 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     5641 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     5701 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     5761 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     5821 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     5881 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     5941 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
//
