LOCUS       sequence-160365-        6309 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  human Doublecortin, N-terminal 6-His tag, C-terminal EGFP-Strep-tag 
            II.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6309)
  AUTHORS   Bechstedt S, Brouhard GJ
  TITLE     Doublecortin recognizes the 13-protofilament microtubule 
            cooperatively and tracks microtubule ends.
  JOURNAL   Dev Cell. 2012 Jul 17;23(1):181-92. doi: 
            10.1016/j.devcel.2012.05.006. Epub 2012 Jun 21.
  PUBMED    22727374
REFERENCE   2  (bases 1 to 6309)
  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: 
            "10.1016/j.devcel.2012.05.006"; journalName: "Dev Cell"; date: 
            "2012-07-17- 17"; volume: "23"; issue: "1"; pages: "181-92"
COMMENT     Sequence Label: hsDCX-EGFP-pHAT
FEATURES             Location/Qualifiers
     source          1..6309
                     /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    19..43
                     /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             56..78
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             116..139
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(1277..1279,1280..1282,1283..1993)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1277..1279,1280..1282,1283..1993)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1322..1343)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1583..1602)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1930..1951
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2012..2035
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(2051..2069)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2052..2069)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2127..2145)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2141..2188
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2424..2445)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    2564..2585
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(2598..3680)
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3642..3661
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3681..3758)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     3904..3923
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4079..4098)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4198..4220
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4258..4276)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4344..4448
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4449..4517,4518..5309)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCHTLLSRIDAGQEQLGRRARYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5480..6068
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5969..5988
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6222..6239
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagca ataattttgt
       61 ttaactttaa gaaggagata tatccatgag tcatcaccat caccatcact ccatgctgga
      121 agttctgttc caggggccca ccatgggtcg acagctgagg ggagctatgg aacttgattt
      181 tggacacttt gacgaaagag ataagacatc caggaacatg cgaggctccc ggatgaatgg
      241 gttgcctagc cccactcaca gcgcccactg tagcttctac cgaaccagaa ccttgcaggc
      301 actgagtaat gagaagaaag ccaagaaggt acgtttctac cgcaatgggg accgctactt
      361 caaggggatt gtgtacgctg tgtcctctga ccgttttcgc agctttgacg ccttgctggc
      421 tgacctgacg cgatctctgt ccgacaacat caacctgcct cagggagtgc gttacattta
      481 caccattgat ggatccagga agatcggaag catggatgaa ctggaggaag gggaaagcta
      541 tgtctgttcc tcagacaact tctttaaaaa ggtggagtac accaagaatg tcaatcccaa
      601 ctggtctgtc aacgtaaaaa catctgccaa tatgaaagcc ccccagtctt tggctagcag
      661 caacagtgca caggccaggg agaacaagga ctttgtgcgc cccaagctgg ttaccatcat
      721 ccgcagtggg gtgaagcctc ggaaggctgt gcgtgtgctt ctgaacaaga agacagccca
      781 ctcttttgag caagtcctca ctgatatcac agaagccatc aaactggaga ccggggttgt
      841 caaaaaactc tacactctgg atggaaaaca ggtaacttgt ctccatgatt tctttggtga
      901 tgatgatgtg tttattgcct gtggtcctga aaaatttcgc tatgctcagg atgatttttc
      961 tctggatgaa aatgaatgcc gagtcatgaa gggaaaccca tcagccacag ctggcccaaa
     1021 ggcatcccca acacctcaga agacttcagc caagagccct ggtcctatgc gccgaagcaa
     1081 gtctccagct gactcaggta acgaccaaga cgcaaacgga acctccagca gccagctctc
     1141 tacccccaag tctaagcagt ctcccatctc tacgcccacc agtcctggca gcctccggaa
     1201 gcacaaggac ctgtacctgc ctctgtcctt ggatgactcg gactcgcttg gtgattccat
     1261 gagttcccct cagcaaatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     1321 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     1381 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1441 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     1501 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1561 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1621 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1681 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1741 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1801 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1861 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     1921 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1981 cgagctgtac aagggatccg cggccgcggc ctggagccac ccgcagttcg aaaaatgaaa
     2041 gcttgagtat tctatagtgt cacctaaatc ccagcttgat ccggctgcta acaaagcccg
     2101 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     2161 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gataacctgg
     2221 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     2281 gaatggaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatatt tgttaaatca
     2341 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2401 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     2461 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cagcttgcat
     2521 gcctgcaggt cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     2581 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     2641 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     2701 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     2761 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     2821 gttgacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     2881 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     2941 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     3001 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     3061 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     3121 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     3181 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     3241 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     3301 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     3361 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     3421 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     3481 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     3541 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     3601 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     3661 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     3721 gccataccgc gaaaggtttt gcaccattcg atggtgtcgg gtaccgagct cgaatttacg
     3781 tgaaccatca cccaaatcaa gttttttgcg gtcgaggtgc cgtaaagctc taaatcggaa
     3841 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     3901 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     3961 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cgcctgatgc
     4021 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta
     4081 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     4141 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4201 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     4261 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     4321 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     4381 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     4441 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     4501 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     4561 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     4621 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtcatacac
     4681 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgggcgcgg tattctcaga
     4741 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     4801 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     4861 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     4921 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     4981 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     5041 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     5101 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     5161 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     5221 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     5281 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     5341 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     5401 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5461 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5521 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5581 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     5641 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5701 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5761 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5821 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5881 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5941 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6001 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6061 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6121 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     6181 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     6241 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     6301 gcagctgat
//
