LOCUS       sequence-213762-        5733 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses tDarpp in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5733)
  AUTHORS   Momand J, Magdziarz P, Feng Y, Jiang D, Parga E, Celis A, Denny E, 
            Wang X, Phillips ML, Monterroso E, Kane SE, Zhou F
  TITLE     t-Darpp is an elongated monomer that binds calcium and is 
            phosphorylated by cyclin-dependent kinases 1 and 5.
  JOURNAL   FEBS Open Bio. 2017 Aug 29;7(9):1328-1337. doi: 
            10.1002/2211-5463.12269. eCollection 2017 Sep.
  PUBMED    28904862
REFERENCE   2  (bases 1 to 5733)
  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: 
            "10.1002/2211-5463.12269"; journalName: "FEBS Open Bio"; date: 
            "2017-08-29- 29"; volume: "7"; issue: "9"; pages: "1328-1337"
COMMENT     Sequence Label: p28atDarpp
FEATURES             Location/Qualifiers
     source          1..5733
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    14..38
                     /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             53..75
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             592..609
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(662..680)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      676..723
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      760..1215
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(847..866)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1057..1078
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1307..2122)
                     /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(1307..2122)
                     /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     2030..2049
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2114..2133
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2244..2832
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2733..2752
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2986..3003
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3018..3157
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3083..3102)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3243..3265
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3259..3450)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4259..5341)
                     /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     5303..5322
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5342..5419)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5625..5644
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5728..5733,1..14)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5728..5733,1..13)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
       61 actttaagaa ggagatatac catggtgttc cggctctcag agcactcctc accagaggag
      121 gaagcctccc cccaccagag agcctcagga gaggggcacc atctcaagtc gaagagaccc
      181 aacccctgtg cctacacacc accttcgctg aaagctgtgc agcgcattgc tgagtctcac
      241 ctgcagtcta tcagcaattt gaatgagaac caggcctcag aggaggagga tgagctgggg
      301 gagcttcggg agctgggtta tccaagagag gaagatgagg aggaagagga ggatgatgaa
      361 gaagaggaag aagaagagga cagccaggct gaagtcctga aggtcatcag gcagtctgct
      421 gggcaaaaga caacctgtgg ccagggtctg gaagggccct gggagcgccc accccctctg
      481 gatgagtccg agagagatgg aggctctgag gaccaagtgg aagacccagc actaagtgag
      541 cctggggagg aacctcagcg cccttccccc tctgagcctg gcacactcga gcaccaccac
      601 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
      661 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
      721 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
      781 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
      841 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
      901 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
      961 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1021 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1081 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1141 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1201 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1261 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     1321 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     1381 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     1441 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     1501 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     1561 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     1621 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     1681 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     1741 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     1801 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     1861 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     1921 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     1981 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     2041 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     2101 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     2161 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2221 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2281 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2341 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     2401 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2461 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2521 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     2581 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     2641 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     2701 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     2761 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     2821 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     2881 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     2941 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3001 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3061 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     3121 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     3181 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3241 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     3301 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     3361 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     3421 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     3481 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     3541 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     3601 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     3661 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     3721 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     3781 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     3841 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     3901 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     3961 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     4021 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     4081 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     4141 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     4201 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     4261 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4321 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     4381 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     4441 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     4501 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     4561 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     4621 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     4681 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     4741 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     4801 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     4861 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     4921 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     4981 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     5041 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     5101 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     5161 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     5221 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     5281 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     5341 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     5401 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     5461 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     5521 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     5581 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     5641 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     5701 aggatcgaga tctcgatccc gcgaaattaa tac
//
