LOCUS       sequence-333267-        4125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway middle entry vector that encodes mCD8mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4125)
  AUTHORS   Chen J, Poskanzer KE, Freeman MR, Monk KR
  TITLE     Live-imaging of astrocyte morphogenesis and function in zebrafish 
            neural circuits.
  JOURNAL   Nat Neurosci. 2020 Oct;23(10):1297-1306. doi: 
            10.1038/s41593-020-0703-x. Epub 2020 Sep 7.
  PUBMED    32895565
REFERENCE   2  (bases 1 to 4125)
  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: 
            "10.1038/s41593-020-0703-x"; journalName: "Nat Neurosci"; date: 
            "2020-10"; volume: "23"; issue: "10"; pages: "1297-1306"
COMMENT     Sequence Label: pME-mCD8mCherry
FEATURES             Location/Qualifiers
     source          1..4125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             182..892
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(327..345)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     595..614
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(935..955)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(935..953)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(982..1081)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1099..1117)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1122..1138)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1122..1138)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1240..1259)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1251..2060
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1318..1337)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2207..2795
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2696..2715
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2949..2966
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3125..3152)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3244..3330)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3379..3401
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3394..3410
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3426..3525
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     3527..3543
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3553..3571
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     3596..3612
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     3597..3613
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(3638..4125,1..175)
                     /codon_start=1
                     /product="mouse lymphocyte antigen CD8 alpha chain"
                     /label=mCD8
                     /translation="MASPLTRFLSLNLLLLGESIILGSGEAKPQAPELRIFPKKMDAEL
                     GQKVDLVCEVLGSVSQGCSWLFQNSSSKLPQPTFVVYMASSHNKITWDEKLNSSKLFSA
                     MRDTNNKYVLTLNKFSKENEGYYFCSVISNSVMYFSSVVPVLQKVNSTTTKPVLRTPSP
                     VHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHS"
ORIGIN
        1 tgtgcaccct accgggacat ctcagcccca gagaccagaa gattgtcggc cccgtggctc
       61 agtgaagggg accggattgg acttcgcctg tgatatttac atctgggcac ccttggccgg
      121 aatctgcgtg gcccttctgc tgtccttgat catcactctc atctgctacc acagcgctag
      181 catggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa
      241 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
      301 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
      361 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
      421 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
      481 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
      541 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
      601 aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
      661 cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc
      721 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
      781 caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
      841 acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt aatctagagc
      901 ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct
      961 tggcgtaatc atggtcatag cacccagctt tcttgtacaa agttggcatt ataagaaagc
     1021 attgcttatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt
     1081 gccatccagc tgatatcccc tatagtgagt cgtattacat ggtcatagct gtttcctggc
     1141 agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat
     1201 catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata
     1261 ttcaacggga aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
     1321 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga
     1381 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     1441 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     1501 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc ggaaaaacag
     1561 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     1621 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     1681 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     1741 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt
     1801 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     1861 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
     1921 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac
     1981 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga
     2041 tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg cagagcatta
     2101 cgctgacttg acgggacggc gcaagctcat gaccaaaatc ccttaacgtg agttacgcgt
     2161 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2221 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2281 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2341 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2401 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2461 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2521 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2581 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2641 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2701 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2761 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     2821 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     2881 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     2941 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     3001 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     3061 cgggcagtga gcgcaacgca attaatacgc gtaccgctag ccaggaagag tttgtagaaa
     3121 cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt tgatgcctgg cagtttatgg
     3181 cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca acgttcaaat ccgctcccgg
     3241 cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca
     3301 gtcttccgac tgagcctttc gttttatttg atgcctggca gttccctact ctcgcgttaa
     3361 cgctagcatg gatgttttcc cagtcacgac gttgtaaaac gacggccagt cttaagctcg
     3421 ggccccaaat aatgatttta ttttgactga tagtgacctg ttcgttgcaa caaattgatg
     3481 agcaatgctt ttttataatg ccaactttgt acaaaaaagc aggcttgtaa aacgacggcc
     3541 agtgagcgcg cgtaatacga ctcactatag ggcgaattgg gtaccgggcc ccccctcgag
     3601 gtcgacggta tcgataagct tgatgcgcgg taccaaaatg gcctcaccgt tgacccgctt
     3661 tctgtcgctg aacctgctgc tgctgggtga gtcgattatc ctggggagtg gagaagctaa
     3721 gccacaggca cccgaactcc gaatctttcc aaagaaaatg gacgccgaac ttggtcagaa
     3781 ggtggacctg gtatgtgaag tgttggggtc cgtttcgcaa ggatgctctt ggctcttcca
     3841 gaactccagc tccaaactcc cccagcccac cttcgttgtc tatatggctt catcccacaa
     3901 caagataacg tgggacgaga agctcaattc gtcgaaactg ttttctgcca tgagggacac
     3961 gaataataag tacgttctca ccctgaacaa gttcagcaag gaaaacgaag gctactattt
     4021 ctgctcagtc atcagcaact cggtgatgta cttcagttct gtcgtgccag tccttcagaa
     4081 agtgaactct actactacca agccagtgct gcgaactccc tcacc
//
