LOCUS       sequence-124753-        6774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of FLAG-and 6xHis-tagged human DND1 protein 
            with defective RRM (Y102A). Codons are optimized for bacteria 
            expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6774)
  AUTHORS   Yamaji M, Jishage M, Meyer C, Suryawanshi H, Der E, Yamaji M, Garzia
            A, Morozov P, Manickavel S, McFarland HL, Roeder RG, Hafner M, 
            Tuschl T
  TITLE     DND1 maintains germline stem cells via recruitment of the CCR4-NOT 
            complex to target mRNAs.
  JOURNAL   Nature. 2017 Mar 23;543(7646):568-572. doi: 10.1038/nature21690. 
            Epub 2017 Mar 15.
  PUBMED    28297718
REFERENCE   2  (bases 1 to 6774)
  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"; doi: 
            "10.1038/nature21690"; journalName: "Nature"; date: "2017-03-23- 
            23"; volume: "543"; issue: "7646"; pages: "568-572"
COMMENT     Sequence Label: pET11d-FHis-DND1-Y102A
FEATURES             Location/Qualifiers
     source          1..6774
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             91..114
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             139..156
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1272..1290)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1286..1333
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1508..1513,1514..1530,1531..1536))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1563..1581)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1649..1753
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1754..1822,1823..2614)
                     /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(1972..1991)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2785..3373
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3274..3293
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3527..3544
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3559..3701
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3627..3646)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3787..3809
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3803..3994)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5306..6388)
                     /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     6350..6369
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6389..6466)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6672..6691
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg gactacaaag acgatgacga taaagcaaga
      121 agagcatctg tgcatatgca tcatcaccat catcacggct gccagtcgaa acgcgattgc
      181 gaattgtggt gcgaacgtgt taaccccgaa aataaagcgg ccctcgaagc atgggtgcgc
      241 gaaacgggca ttcgtttagt ccaggttaac ggtcagcgca aatatggtgg tcctcctccg
      301 ggttgggtag ggtctcctcc gccggcgggc agcgaagtgt tcattggccg cctcccgcaa
      361 gatgtttatg agcaccaact gattccgctg ttccaacgtg tgggtcgtct ctacgaattc
      421 cggctgatga tgacgttttc tggcctgaat cgtgggtttg ctgctgcacg ttattctagc
      481 cgtcgtggtg cccaggcggc tatcgctacg ctgcataatc acccgctgcg tccctcctgt
      541 cctctcttag tctgccgtag cactgaaaag tgtgaattgt ctgtggatgg tcttccgcca
      601 aacttgaccc gttcggcgct gttattggca ctgcaaccct taggcccggg cctccaggaa
      661 gcacgcctgc ttccgagccc cggcccggct cctgggcaga ttgcactcct caaattttca
      721 tcacatcgtg cggcggcaat ggccaaaaaa gcattggtgg agggccaatc tcacttgtgt
      781 ggtgaacagg tcgcagtgga gtggctgaaa cctgatctga aacagcggtt gcgtcagcaa
      841 ttggtgggac cgttcttgcg ttcaccgcag ccggagggtt cccagcttgc actggcccgg
      901 gacaaacttg gtttccaggg tgcgcgtgca acattacagc ttctctgtca gcgtatgaag
      961 ctgggctccc ctgtctttct gacgaaatgc ctgggcatcg ggccggcagg ctggcatcgt
     1021 ttctggtatc aagtcgtgat tccgggccac ccggtcccgt tcagcgggtt aatttgggtt
     1081 gttctgacgc ttgacggccg cgatggccat gaggtggcca aagatgccgt ttcggttcgg
     1141 ctgctgcagg ccctcagtga gagtggggcg aacctgctct ggtcggcagg cgctgaagct
     1201 ggtacgatgg taaaacagtg aggatccggc tgctaacaaa gcccgaaagg aagctgagtt
     1261 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     1321 gaggggtttt ttgctgaaag gaggaactat atccggatat cccgcaagag gcccggcagt
     1381 accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag gatgacgatg
     1441 agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa
     1501 ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattctt gaagacgaaa
     1561 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     1621 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1681 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     1741 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     1801 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     1861 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     1921 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     1981 cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc
     2041 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     2101 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     2161 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     2221 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     2281 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     2341 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     2401 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     2461 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     2521 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     2581 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     2641 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     2701 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2761 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2821 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2881 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     2941 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3001 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3061 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3121 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3181 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3241 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3301 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3361 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3421 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3481 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3541 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3601 acaccgcata tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3661 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     3721 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3781 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     3841 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     3901 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     3961 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     4021 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     4081 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     4141 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     4201 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     4261 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     4321 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4381 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4441 gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc
     4501 tgctggagat ggcggacgcg atggatatgt tctgccaagg gttggtttgc gcattcacag
     4561 ttctccgcaa gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc
     4621 gccggcttcc attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag
     4681 gcagacaagg tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga
     4741 ggcggcataa atcgccgtga cgatcagcgg tccagtgatc gaagttaggc tggtaagagc
     4801 cgcgagcgat ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc
     4861 ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat
     4921 ccagcctcgc gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc
     4981 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     5041 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     5101 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     5161 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     5221 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     5281 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5341 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     5401 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     5461 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     5521 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     5581 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     5641 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     5701 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     5761 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     5821 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     5881 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5941 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     6001 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     6061 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     6121 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     6181 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     6241 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     6301 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     6361 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     6421 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     6481 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     6541 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     6601 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     6661 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     6721 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaat
//
