LOCUS       sequence-313970-        6581 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To tag endogenously and c-terminally mouse Mecp2 with two reporters:
            NanoLuciferase and TdTomato. It contains 2 homology arms from a 
            castaneus background surrounding the STOP codon..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6581)
  AUTHORS   Mira-Bontenbal H, Tan B, Gontan C, Goossens S, Boers RG, Boers JB, 
            Dupont C, van Royen ME, IJcken WFJ, French P, Bedalov A, Gribnau J
  TITLE     Genetic and epigenetic determinants of reactivation of Mecp2 and the
            inactive X chromosome in neural stem cells.
  JOURNAL   Stem Cell Reports. 2022 Jan 29. pii: S2213-6711(22)00052-2. doi: 
            10.1016/j.stemcr.2022.01.008.
  PUBMED    35148843
REFERENCE   2  (bases 1 to 6581)
  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: "Stem Cell 
            Reports. 2022 Jan 29. pii: S2213-6711(22)00052-2. doi: 
            10.1016/j.stemcr.2022.01.008."
COMMENT     Sequence Label: TOPO mMecp2-NLucTom
FEATURES             Location/Qualifiers
     source          1..6581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             456..476
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             819..1331
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             1347..1403
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1419..1439
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1440..2870
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     primer_bind     complement(3469..3488)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3470..3488)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3495..3512)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3495..3511)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3504..3526)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3649..3951
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     3830..3849
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             4300..5094
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4300..5094
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4354..4373)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4964..4983
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      5294..5303
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5300..5674
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      5812..6400
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6301..6320
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6554..6571
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      301 tcgaggtcaa ggaagtggtg aagcccctgc tggtgtccac ccttggtgag aaaagcggga
      361 agggactgaa gacctgcaag agccctgggc gtaaaagcaa ggagagcagc cccaaggggc
      421 gcagcagcag tgcctcctcc ccacctaaga aggagcacca tcatcaccac catcactcag
      481 agtccacaaa ggcccccatg ccactgctcc catccccacc cccacctgag cctgagagct
      541 ctgaggaccc catcagcccc cctgagcctc aggacttgag cagcagcatc tgcaaagaag
      601 agaagatgcc ccgaggaggc tcactggaaa gcgatggctg ccccaaggag ccagctaaga
      661 ctcagcctat ggtcgccgcc actaccacag ttgcagaaaa gtacaaacac cgaggggagg
      721 gagagcgcaa agacattgtt tcatcttcca tgccaaggcc aaacagagag gagcctgtgg
      781 acagccggac gcccgtgacc gagagagtta gcggatccat ggtcttcaca ctcgaagatt
      841 tcgttgggga ctggcgacag acagccggct acaacctgga ccaagtcctt gaacagggag
      901 gtgtgtccag tttgtttcag aatctcgggg tgtccgtaac tccgatccaa aggattgtcc
      961 tgagcggtga aaatgggctg aagatcgaca tccatgtcat catcccgtat gaaggtctga
     1021 gcggcgacca aatgggccag atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc
     1081 atcactttaa ggtgatcctg cactatggca cactggtaat cgacggggtt acgccgaaca
     1141 tgatcgacta tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca
     1201 ctgtaacagg gaccctgtgg aacggcaaca aaattatcga cgagcgcctg atcaaccccg
     1261 acggctccct gctgttccga gtaaccatca acggagtgac cggctggcgg ctgtgcgaac
     1321 gcattctggc ggaattcgga agcggagcta ctaacttcag cctgctgaag caggctggag
     1381 acgtggagga gaaccctgga cctgctagca tgactgctcc aaagaagaag cgtaaggtaa
     1441 tggtgagcaa gggcgaggag gtcatcaaag agttcatgcg cttcaaggtg cgcatggagg
     1501 gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     1561 gcacccagac cgccaagctg aaggtgacca agggcggccc cctgcccttc gcctgggaca
     1621 tcctgtcccc ccagttcatg tacggctcca aggcgtacgt gaagcacccc gccgacatcc
     1681 ccgattacaa gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     1741 aggacggcgg tctggtgacc gtgacccagg actcctccct gcaggacggc acgctgatct
     1801 acaaggtgaa gatgcgcggc accaacttcc cccccgacgg ccccgtaatg cagaagaaga
     1861 ccatgggctg ggaggcctcc accgagcgcc tgtacccccg cgacggcgtg ctgaagggcg
     1921 agatccacca ggccctgaag ctgaaggacg gcggccacta cctggtggag ttcaagacca
     1981 tctacatggc caagaagccc gtgcaactgc ccggctacta ctacgtggac accaagctgg
     2041 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc tccgagggcc
     2101 gccaccacct gttcctgggg catggcaccg gcagcaccgg cagcggcagc tccggcaccg
     2161 cctcctccga ggacaacaac atggccgtca tcaaagagtt catgcgcttc aaggtgcgca
     2221 tggagggctc catgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
     2281 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg cggccccctg cccttcgcct
     2341 gggacatcct gtccccccag ttcatgtacg gctccaaggc gtacgtgaag caccccgccg
     2401 acatccccga ttacaagaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga
     2461 acttcgagga cggcggtctg gtgaccgtga cccaggactc ctccctgcag gacggcacgc
     2521 tgatctacaa ggtgaagatg cgcggcacca acttcccccc cgacggcccc gtaatgcaga
     2581 agaagaccat gggctgggag gcctccaccg agcgcctgta cccccgcgac ggcgtgctga
     2641 agggcgagat ccaccaggcc ctgaagctga aggacggcgg ccactacctg gtggagttca
     2701 agaccatcta catggccaag aagcccgtgc aactgcccgg ctactactac gtggacacca
     2761 agctggacat cacctcccac aacgaggact acaccatcgt ggaacagtac gagcgctccg
     2821 agggccgcca ccacctgttc ctgtacggca tggacgagct gtacaagtaa actagtgcaa
     2881 agcaaaccaa caagaataaa ggcagctgtt gtctcttctc cttatgggta gggctctgac
     2941 aaagcttccc gattaactga aataaaaaat attttttttt ctttcagtaa acttagagtt
     3001 tcgtggcttc ggggtgggag tagttggagc attgggatgt ctttcttacc gacaagcaca
     3061 gtcaggttga agacctaacc agggccagaa gtagctttgc acttttctaa actaggctcc
     3121 ttcaacaagg cttgctgcag atactactga ccagacaagc tgttgaccag gcactccccc
     3181 caacaatatc ctccctcttc cccccccacc ccacccccgc cccgtgtgct cgttagggca
     3241 attgagagga cactcccatt tttggtgcca ttgatgccct gtccataata gcttccctga
     3301 cttttacacc accccaactc ccaatctgaa ggactgggag gtgtgatgca ggagaaacta
     3361 tgggactctt gggagaagac tatggagttg gccagtgatt aaggcccagt aattccaact
     3421 gtggtagcac agatctggct ccacatcaac ccaatcgggc ccaattcgcc ctatagtgag
     3481 tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     3541 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     3601 gcccgcaccg atcgcccttc ccaacagttg cgcagcctat acgtacggca gtttaaggtt
     3661 tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt
     3721 gacacgccgg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa
     3781 gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc
     3841 accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac
     3901 cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc
     3961 atgagattat caaaaaggat cttcacctag atccttttca cgtagaaagc cagtccgcag
     4021 aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca
     4081 agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg
     4141 gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg
     4201 aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga
     4261 tcaagctctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     4321 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     4381 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     4441 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     4501 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     4561 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     4621 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     4681 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4741 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4801 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     4861 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4921 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4981 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     5041 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaattatt
     5101 aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     5161 cgcatcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5221 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     5281 gcacgtgagg agggccacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
     5341 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
     5401 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
     5461 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
     5521 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
     5581 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
     5641 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctaaaacttc atttttaatt
     5701 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5761 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5821 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5881 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5941 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6001 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6061 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6121 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6181 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6241 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6301 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6361 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6421 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     6481 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     6541 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa g
//
