LOCUS       sequence-170472-        4365 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Middle entry vector for inserting promotors for tissue specific 
            expression of NLS-tdMCP-eGFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4365)
  AUTHORS   Campbell PD, Chao JA, Singer RH, Marlow FL
  TITLE     Dynamic visualization of transcription and RNA subcellular 
            localization in zebrafish.
  JOURNAL   Development. 2015 Apr 1;142(7):1368-74. doi: 10.1242/dev.118968. 
            Epub 2015 Mar 10.
  PUBMED    25758462
REFERENCE   2  (bases 1 to 4365)
  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: 
            "10.1242/dev.118968"; journalName: "Development"; date: "2015-04-1- 
            1"; volume: "142"; issue: "7"; pages: "1368-74"
COMMENT     Sequence Label: pME-NLS-tdMCP-eGFP
FEATURES             Location/Qualifiers
     source          1..4365
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(321..323,324..326,327..1040)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(321..323,324..326,327..1037)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(366..387)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(627..646)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     974..995
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1066..1165)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1182..1201)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1183..1201)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1206..1222)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1206..1222)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1653..2444
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      2537..3125
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3026..3045
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3279..3296
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3455..3482)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3574..3660)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3709..3731
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3723..3740
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3724..3740
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3756..3855
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3888..3908
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3912..3938
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             3912..3938
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3939..3950
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
ORIGIN
        1 gaactggcga cgtgactgtc gccccaagca acttcgctaa cgggatcgct gaatggatca
       61 gctctaactc gcgttcacag gcttacaaag taacctgtag cgttcgtcag agctctgcgc
      121 agaatcgcaa atacaccatc aaagtcgagg tgcctaaagg cgcctggcgt tcgtacttaa
      181 atatggaact aaccattcca attttcgcca cgaattccga ctgcgagctt attgttaagg
      241 caatgcaagg tctcctaaaa gatggaaacc cgattccctc agcaatcgca gcaaactccg
      301 gcatctacgc ggattctaga atggtgagca agggcgagga gctgttcacc ggggtggtgc
      361 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
      421 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
      481 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
      541 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
      601 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
      661 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
      721 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
      781 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
      841 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
      901 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
      961 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     1021 tggacgagct gtacaagtaa accggttctc tcaagggcga attcgaccca gctttcttgt
     1081 acaaagttgg cattataaaa aataattgct catcaatttg ttgcaacgaa caggtcacta
     1141 tcagtcaaaa taaaatcatt atttgccatc cagctgatat cccctatagt gagtcgtatt
     1201 acatggtcat agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca
     1261 ttgcacaaga taaaaatata tcatcatgcc tcctctagac cagccaggac agaaatgcct
     1321 cgacttcgct gctacccaag gttgccgggt gacgcacacc gtggaaacgg atgaaggcac
     1381 gaacccagtg gacataagcc tgttcggttc gtaagctgta atgcaagtag cgtatgcgct
     1441 cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt
     1501 tcatggcttg ttatgactgt ttttttgggg tacagtctat gcctcgggca tccaagcagc
     1561 aagcgcgtta cgccgtgggt cgatgtttga tgttatggag cagcaacgat gttacgcagc
     1621 agggcagtcg ccctaaaaca aagttaaaca ttatgaggga agcggtgatc gccgaagtat
     1681 cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg
     1741 ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt
     1801 tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc
     1861 ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca
     1921 ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg
     1981 gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg
     2041 atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg
     2101 cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct
     2161 taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt
     2221 tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg
     2281 actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctagacagg
     2341 cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg
     2401 tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataaccctcg agccacccat
     2461 gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa
     2521 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2581 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2641 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     2701 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2761 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2821 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2881 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2941 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3001 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3061 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3121 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3181 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3241 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3301 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     3361 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatacgc
     3421 gtaccgctag ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt
     3481 ctgcttagtt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg
     3541 ttgcttcaca acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac
     3601 cgacaaacaa cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg
     3661 atgcctggca gttccctact ctcgcgttaa cgctagcatg gatgttttcc cagtcacgac
     3721 gttgtaaaac gacggccagt cttaagctcg ggccccaaat aatgatttta ttttgactga
     3781 tagtgacctg ttcgttgcaa caaattgatg agcaatgctt ttttataatg ccaactttgt
     3841 acaaaaaagc aggctccgaa ttcgcccttg agagactcga gatgggccca aaaaagaaaa
     3901 gaaaagttgg ctacccctac gacgtgcccg actacgccat cgaaggccgc catatgctag
     3961 ccgttaaaat ggcttctaac tttactcagt tcgttctcgt cgacaatggc ggaactggcg
     4021 acgtgactgt cgccccaagc aacttcgcta acgggatcgc tgaatggatc agctctaact
     4081 cgcgttcaca ggcttacaaa gtaacctgta gcgttcgtca gagctctgcg cagaatcgca
     4141 aatacaccat caaagtcgag gtgcctaaag gcgcctggcg ttcgtactta aatatggaac
     4201 taaccattcc aattttcgcc acgaattccg actgcgagct tattgttaag gcaatgcaag
     4261 gtctcctaaa agatggaaac ccgattccct cagcaatcgc agcaaactcc ggcatctacg
     4321 ccatggcttc taactttact cagttcgttc tcgtcgacaa tggcg
//
