LOCUS       sequence-252344-        4832 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  a mammalian expression backbone vector for humanized 
            mNeonGreen-3xFlag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4832)
  AUTHORS   Tanida-Miyake E, Koike M, Uchiyama Y, Tanida I
  TITLE     Optimization of mNeonGreen for Homo sapiens increases its 
            fluorescent intensity in mammalian cells.
  JOURNAL   PLoS One. 2018 Jan 17;13(1):e0191108. doi: 
            10.1371/journal.pone.0191108. eCollection 2018.
  PUBMED    29342181
REFERENCE   2  (bases 1 to 4832)
  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: "PLoS One.";
            date: "2018-01-17"; volume: "13(1):e0191108. doi"; pages: " 
            10.1371/journal.pone.0191108. eCollection 2018"
COMMENT     Sequence Label: pmNeonGreenHO-3xFLAG-G
FEATURES             Location/Qualifiers
     source          1..4832
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      537..546
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1278..1301
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1314..1379
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    1380..1436
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    1560..1681
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1597..1616)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1651..1670
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1688..2143)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1825..1846)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2037..2056
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2170..2274
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2276..2633
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2276..2467
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2299..2319)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2484..2619
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2668..3462
                     /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             2668..3462
                     /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(2722..2741)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3332..3351
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3650..3669)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3694..3741
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4070..4658
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4559..4578
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcca
      541 ccatggtcag caaaggcgaa gaagacaaca tggcatcact gccagcgaca cacgaacttc
      601 atattttcgg ctccatcaat ggtgtggact tcgatatggt tggacagggt actgggaacc
      661 ctaacgatgg atatgaggag ttgaacttga agagtaccaa gggcgatctt cagttttcac
      721 cttggattct cgtacctcat ataggttatg gctttcatca atatctgccc tatcctgacg
      781 gtatgtcacc tttccaagca gctatggttg acgggagcgg ttatcaggtt caccggacaa
      841 tgcagtttga agatggagcc tctttgaccg taaattatcg gtatacttat gagggcagcc
      901 atattaaagg cgaggctcaa gttaagggga ctgggttccc cgcggatggt cccgtaatga
      961 caaacagtct gacggctgca gactggtgcc gctctaagaa gacatatcca aatgacaaga
     1021 cgattattag cactttcaag tggtcataca ctacaggaaa tggtaaacgc tataggagca
     1081 ctgcccggac cacgtatact ttcgcgaagc cgatggcggc aaattatttg aaaaaccaac
     1141 cgatgtatgt gttcagaaag acagaactca agcattctaa aactgaactg aacttcaaag
     1201 agtggcagaa agcctttact gatgtaatgg gcatggacga actctataag tccggaggtg
     1261 gatcgggagg tggatcgtgg agccacccgc agttcgaaaa aactagtcgg gctgactaca
     1321 aagaccatga cggtgattat aaagatcatg acatcgacta caaggatgac gatgacaaga
     1381 gatctcgagc tcaagcttcg aattctgcag tcgacggtac cgcgggcccg ggatccaccg
     1441 gatctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     1501 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     1561 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     1621 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     1681 aacgcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     1741 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     1801 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     1861 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     1921 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     1981 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     2041 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     2101 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg
     2161 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2221 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag
     2281 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     2341 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     2401 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     2461 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     2521 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     2581 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat
     2641 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     2701 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     2761 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     2821 gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc
     2881 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     2941 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     3001 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     3061 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     3121 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     3181 gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     3241 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     3301 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     3361 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     3421 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     3481 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     3541 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     3601 gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga
     3661 aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc
     3721 acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg
     3781 ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc
     3841 accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg
     3901 ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat ttttaattta
     3961 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4021 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4081 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4141 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4201 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4261 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4321 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4381 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4441 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4501 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4561 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4621 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4681 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4741 attctgtgga taaccgtatt accgccatgc attagttatt aatagtaatc aattacgggg
     4801 tcattagttc atagcccata tatggagttc cg
//
