LOCUS       sequence-462712-        6885 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression of GNL2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6885)
  TITLE     Iuchi Lab GNL2 Plasmids 2025
REFERENCE   2  (bases 1 to 6885)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pGNL2-#1
FEATURES             Location/Qualifiers
     source          1..6885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3673..3794
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3710..3729)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3764..3783
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3801..4256)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     4150..4169
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4283..4387
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4389..4746
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4389..4580
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4412..4432)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4597..4732
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4781..5575
                     /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             4781..5575
                     /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(4835..4854)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5445..5464
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5763..5782)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5807..5854
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6183..6771
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6672..6691
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggccggac tcagatctcg agctcaagct tcgaattcat ggtgaagccc
     1381 aagtacaaag gacggagcac catcaacccg tccaaggcca gcacaaaccc agatcgagtg
     1441 cagggagcag gaggccaaaa catgagggac cgggccacca tccggcgcct gaatatgtat
     1501 aggcaaaagg agcgcaggaa cagtcgtggt aaaataatta aacccctgca atatcaatca
     1561 acggtggctt ctggcacagt ggcaagagta gagccaaata ttaaatggtt tggaaacaca
     1621 cgtgtgatta agcagtcatc attacaaaaa tttcaagagg aaatggatac agttatgaag
     1681 gatccataca aagttgtcat gaagcaaagc aagttaccaa tgtctcttct ccatgatcga
     1741 atccggcctc ataacttgaa ggtgcacatt cttgatactg aaagttttga aactacattt
     1801 ggccctaagt cacagaggaa acgaccaaac ttatttgcaa gtgatatgca gtctcttatc
     1861 gaaaatgctg aaatgtccac tgagagctat gaccagggca aggatcgtga tttggtaact
     1921 gaagacactg gtgtgagaaa tgaagctcaa gaagagatct ataaaaaggg acagtccaaa
     1981 agaatatggg gtgagctcta caaggtgata gattcatcag atgttgtagt tcaagttctt
     2041 gatgctagag atccaatggg tactcgttcc cctcacattg aaacttacct gaagaaggaa
     2101 aaaccttgga aacacctcat ttttgtactt aacaaatgtg accttgttcc aacctgggca
     2161 acaaaacggt gggttgctgt cctctcccag gattatccaa cacttgcttt ccatgcaagc
     2221 cttactaacc cgtttggcaa gggagcattc attcagcttc tgcggcagtt tggaaagttg
     2281 cacactgaca agaaacagat cagtgttggg ttcattggct atccaaatgt tggcaagagc
     2341 tctgtgataa atacattgcg ttctaagaaa gtttgcaacg tggctcccat tgcaggtgaa
     2401 acaaaggtct ggcagtatat tactttgatg cgtcggatat tcctgattga ctgtccaggt
     2461 gtggtttacc cctctgagga ctccgagaca gacattgtgc taaaaggagt tgttcaagta
     2521 gaaaaaatta agagtcctga agaccacatt ggtgctgtac ttgaacgagc aaagccagaa
     2581 tatatcagca aaacatacaa gattgattct tgggagaatg ctgaggactt tcttgagaag
     2641 ctcgctttcc ggactgggaa gttactaaag ggtggagagc ccgacttgca gactgtgggt
     2701 aagatggtcc tcaatgactg gcagaggggc cggattcctt tctttgtcaa gccacccaat
     2761 gcagagccac ttgtggcccc ccagcttcta ccctcctcat ctttggaagt tgtcccagaa
     2821 gcagcccaga acaatccagg ggaggaagtc acagaaactg caggtgaagg gtcagaatcc
     2881 atcattaagg aagaaacaga agagaacagt cactgtgatg ctaacacaga gatgcagcag
     2941 attctcacac gagttcggca gaactttggt aaaatcaacg tggtgcctca gttttctggg
     3001 gatgacctgg ttcctgtgga ggtgtcagat cttgaggaag agcttgagag cttttctgat
     3061 gaagaggagg aggaacagga gcaacaaaga gatgatgcgg aagagtcttc ctcggagcct
     3121 gaggaggaaa atgtgggaaa cgacaccaaa gccgttatta aagcactgga tgagaagatt
     3181 gccaaatatc agaagtttct agacaaagcc aaagccaaaa agttttcagc agtcagaata
     3241 tccaagggac tgagtgaaaa gatatttgca aaacctgaag aacaaagaaa aacactggaa
     3301 gaagatgtag atgacagagc accttccaaa aagggaaaga agcggaaggc acaaagggaa
     3361 gaggaacagg aacattcaaa taaagctccc agggcgctta catcaaaaga acggaggcga
     3421 gcagtacgac agcaacggcc gaaaaaagtt ggtgtgcgct actatgaaac acacaacgtg
     3481 aaaaatagga acaggaacaa aaagaagacc aatgactcag agggacagaa acacaaacgc
     3541 aaaaaattca gacaaaagca gtaactgatc ataatcagcc ataccacatt tgtagaggtt
     3601 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     3661 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     3721 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     3781 atcaatgtat cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     3841 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     3901 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     3961 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     4021 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     4081 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     4141 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     4201 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     4261 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4321 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4381 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     4441 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4501 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4561 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4621 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4681 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4741 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     4801 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     4861 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     4921 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     4981 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     5041 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     5101 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     5161 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     5221 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     5281 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     5341 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     5401 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     5461 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     5521 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     5581 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     5641 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     5701 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     5761 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     5821 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     5881 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     5941 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     6001 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt
     6061 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6121 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6181 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6241 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6301 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6361 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6421 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6481 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6541 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6601 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6661 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6721 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6781 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6841 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat
//
