LOCUS       sequence-389416-        6523 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Codon-optimized bacterial expression plasmid. Expresses 
            GB1-His6-TEV-A11(2-196)-Strep..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6523)
  AUTHORS   Shihora A, Elias RD, Hammond JA, Ghirlando R, Deshmukh L
  TITLE     ALS Variants of Annexin A11's Proline-Rich Domain Impair Its 
            S100A6-Mediated Fibril Dissolution.
  JOURNAL   ACS Chem Neurosci. 2023 Jul 11. doi: 10.1021/acschemneuro.3c00169.
  PUBMED    37433222
REFERENCE   2  (bases 1 to 6523)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Chem 
            Neurosci. 2023 Jul 11. doi: 10.1021/acschemneuro.3c00169."
COMMENT     Sequence Label: GB1-A11(2-196)-Strep
FEATURES             Location/Qualifiers
     source          1..6523
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     79..98
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     332..349
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    364..503
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(429..448)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     589..611
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(605..796)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2108..3190)
                     /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     3152..3171
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3191..3268)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3474..3493
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3577..3596
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3577..3595
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3596..3620
                     /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)."
     RBS             3635..3657
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3833..3850
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3851..3862
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3902..3919
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3920..3940
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             4523..4546
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(4600..4618)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4614..4661
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4836..4841,4842..4858,4859..4864))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4891..4909)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4977..5081
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5082..5150,5151..5942)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5300..5319)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6113..6523,1..178)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
       61 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
      121 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
      181 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
      241 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
      301 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      361 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
      421 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
      481 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
      541 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
      601 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
      661 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
      721 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
      781 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
      841 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
      901 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
      961 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     1021 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     1081 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     1141 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     1201 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     1261 gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag atggcggacg
     1321 cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc aagaattgat
     1381 tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt
     1441 cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa ggtatagggc
     1501 ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt
     1561 gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg atccttgaag
     1621 ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg cgggcatccc
     1681 gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa
     1741 cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc
     1801 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     1861 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     1921 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     1981 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     2041 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     2101 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     2161 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     2221 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     2281 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     2341 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     2401 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     2461 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     2521 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     2581 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     2641 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     2701 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     2761 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     2821 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     2881 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     2941 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     3001 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     3061 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     3121 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     3181 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     3241 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     3301 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     3361 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     3421 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     3481 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     3541 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     3601 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     3661 acatatgcag tataaactgg cgctgaacgg caaaaccctg aaaggcgaaa ccaccaccga
     3721 agcggtggat gcggcgaccg cggaaaaagt gtttaaacag tatgcgaacg ataacggcgt
     3781 ggatggcgaa tggacctatg atgatgcgac caaaaccttt accgtgaccg aactggtgcc
     3841 gcgcggcagc attgaaggcc gcattcgcga aaacctgaaa gatagcggcc tgtttctgga
     3901 acatcatcat catcatcatg aaaacctgta tttccaatct tatccgggct atccgccgcc
     3961 accgggtggg tatccgccgg cggcaccggg cggtggtccg tggggcggtg cggcgtatcc
     4021 gccgcctccg tctatgccgc cgattggcct ggataacgtg gcgacctatg cgggtcagtt
     4081 taaccaagat tatctgagcg gcatggcggc gaacatgagc ggcacctttg gcggtgcaaa
     4141 catgccgaac ttgtacccgg gcgctccggg tgcggggtac ccgccggtac cacctggtgg
     4201 gtttgggcag ccgccgagcg cgcaacagcc tgttccgccg tacgggatgt acccgccgcc
     4261 gggcggcaac ccaccgagcc gcatgccgtc atatccaccg taccctgggg caccggtccc
     4321 tggtcaaccg atgccaccgc ctgggcaaca accgccgggt gcttacccgg gtcagccgcc
     4381 tgttacgtat cctggtcagc cgccggtgcc gctcccgggt caacagcaac cggtgccatc
     4441 atacccgggc tacccgggca gcggcaccgt aactccggcg gtgccgccga cgcagttcgg
     4501 cagcagaggc accatcaccg actggagcca tccgcagttt gaaaaataag gatccggctg
     4561 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     4621 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     4681 ccggatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc
     4741 cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg
     4801 actgcgttag caatttaact gtgataaact accgcattaa agcttatcga tgataagctg
     4861 tcaaacatga gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta
     4921 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     4981 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5041 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     5101 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5161 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5221 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5281 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     5341 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5401 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5461 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5521 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5581 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     5641 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     5701 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     5761 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     5821 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     5881 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     5941 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     6001 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6061 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6121 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6181 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6241 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6301 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6361 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6421 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6481 aactgagata cctacagcgt gagctatgag aaagcgccac gct
//
