LOCUS       sequence-474429-        4455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR template to insert mEmerald before the stop codon in exon 7 of 
            the human doublecortin (DCX) locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4455)
  AUTHORS   Dema A, Charafeddine RA, van Haren J, Rahgozar S, Viola G, Jacobs 
            KA, Kutys ML, Wittmann T
  TITLE     Doublecortin reinforces microtubules to promote growth cone advance 
            in soft environments.
  JOURNAL   Curr Biol. 2024 Nov 21:S0960-9822(24)01458-1. doi: 
            10.1016/j.cub.2024.10.063.
  PUBMED    39626666
REFERENCE   2  (bases 1 to 4455)
  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: "Curr Biol. 
            2024 Nov 21:S0960-9822(24)01458-1. doi: 10.1016/j.cub.2024.10.063."
COMMENT     Sequence Label: pDCX_mEmerald_CRISPR_HDR
FEATURES             Location/Qualifiers
     source          1..4455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_feature    1004..1060
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     CDS             join(1579..1581,1582..1584,1585..2298)
                     /codon_start=1
                     /product="brightly fluorescent monomeric variant of GFP"
                     /label=mEmerald
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1624..1645)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1885..1904)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2232..2253
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2830..2847)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2830..2846)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2839..2861)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3055..3074)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3174..3196
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3234..3252)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3320..3424
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3425..3493,3494..4285)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3643..3662)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg catgcctgca
     1021 ggtcgactct agaggatccc cgggtaccga gctcgaattc tataacattg ttgaaattcc
     1081 aaaattacag agacaggaaa cagattattt gttgccaggg ctcagggaag aggggagggt
     1141 tggatggagc tctaatgggg tagcacaagg gagccttgtg ttgaaggtac agttctgtat
     1201 cttgactgtg gcgatggtca cacaaatata cacacatgat aaaattgcat aggaatatac
     1261 acacacacac atacacaccc cacacacaaa tgatcacatg ttaaactgca agatggtacc
     1321 attagggaaa attgaatgaa gggtacattg gtacaagggt acctccctgt acattttttc
     1381 aacttcctgt gaatctataa ttattatgtt gtaaaaatta aagtattaat aaaaaaaact
     1441 aaagcagaca ttccagagct caagatatca agaaaaggaa aattaacttt gtctcttctc
     1501 ttcttatagg acctgtacct gcctctgtcc ttggacgatt cagattccct cggcgactca
     1561 atgggttctg gaagtggaat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     1621 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     1681 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1741 cccgtgccct ggcccaccct cgtgaccacc ttgacctacg gcgtgcagtg cttcgcccgc
     1801 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1861 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1921 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1981 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaaggt ctatatcacc
     2041 gccgacaagc agaagaacgg catcaaggtg aacttcaaga cccgccacaa catcgaggac
     2101 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2161 ctgctgcccg acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag
     2221 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     2281 gacgagctgt acaagtaaag gaggggagag tgctcagagt ccagagtaca aatccaagcc
     2341 tatcattgta gtagggtact tctgctcaag tgtccaacag ggctattggt gctttcaagt
     2401 ttttattttg ttgttgttgt tattttgaaa aacacattgt aatatgttgg gtttattttc
     2461 ctgtgatttc tcctctgggc cactgatcca cagttaccaa ttatgagaga tagattgata
     2521 accatccttt ggggcagcat tccagggatg caaaatgtgc tagtccatga cctttcaatg
     2581 gaaagcttag gtgcctgcgt tatatttgcc ctgtctaatt ttgcccatac agtcttcctt
     2641 ctgtagaggg ctgtttacat atacagcact taaaatgttt gtgtgggaaa aaaaaaactc
     2701 attggcagat ccaagaatga caaacacaag tgcccctttt ctctggatct caagaatggt
     2761 ggaggaccct ggaaggacag caaggcagct ccccagcctc actcttcact cctgattgag
     2821 gccgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     2881 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     2941 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
     3001 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
     3061 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     3121 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3181 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     3241 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     3301 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     3361 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3421 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     3481 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     3541 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     3601 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     3661 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     3721 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     3781 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     3841 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     3901 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     3961 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     4021 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     4081 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     4141 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     4201 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     4261 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     4321 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     4381 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     4441 gatcaaagga tcttc
//
