LOCUS       sequence-163625-        4732 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Localization: N1 Cloning Vector, Excitation: 399, Emission: 511.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4732)
  AUTHORS   Ai HW, Hazelwood KL, Davidson MW, Campbell RE
  TITLE     Fluorescent protein FRET pairs for ratiometric imaging of dual 
            biosensors.
  JOURNAL   Nat Methods. 2008 May;5(5):401-3. doi: 10.1038/nmeth.1207.
  PUBMED    18425137
REFERENCE   2  (bases 1 to 4732)
  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/nmeth"; 
            journalName: "Nat Methods"; date: "2008-05"; volume: "5"; issue: 
            "5"; pages: "401-3"
COMMENT     Sequence Label: mT-Sapphire-N1
FEATURES             Location/Qualifiers
     source          1..4732
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        16..319
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        320..523
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     474..494
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    546..626
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      628..637
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(634..636,637..639,640..1350)
                     /codon_start=1
                     /product="strongly enhanced green fluorescent protein 
                     (Kremers et al., 2007)"
                     /label=SGFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVMVFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSIQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(679..700)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(940..959)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1287..1308
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1475..1596
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1512..1531)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1566..1585
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1603..2058)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1740..1761)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1952..1971
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2085..2189
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2191..2548
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2191..2382
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2214..2234)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2399..2534
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2583..3377
                     /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             2583..3377
                     /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(2637..2656)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3247..3266
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3565..3584)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3609..3656
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3985..4573
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4474..4493
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
       61 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      121 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      181 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      241 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      301 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      361 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      421 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      481 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
      541 gatccgctag cgctaccgga ctcagatctc gagctcaagc ttcgaattct gcagtcgacg
      601 gtaccgcggg cccgggatcc accggtcgcc accatggtga gcaagggcga ggagctgttc
      661 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
      721 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
      781 accaccggca agctgcccgt gccctggccc accctcgtga ccaccttctc ctacggcgtg
      841 atggtgttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
      901 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
      961 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1021 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaactt caacagccac
     1081 aacgtctata tcatggccga caagcagaag aacggcatca aggccaactt caagatccgc
     1141 cacaacatcg aggacggcgg cgtgcagctc gccgaccact accagcagaa cacccccatc
     1201 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcatccagtc caagctgagc
     1261 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1321 atcactctgg gcatggacga gctgtacaag taagcggccg cgactctaga tcataatcag
     1381 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     1441 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     1501 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     1561 tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata
     1621 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     1681 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     1741 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     1801 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     1861 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     1921 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     1981 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     2041 cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     2101 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     2161 tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt
     2221 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     2281 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     2341 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     2401 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     2461 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     2521 cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc
     2581 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     2641 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     2701 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     2761 tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     2821 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     2881 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     2941 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     3001 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     3061 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg
     3121 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     3181 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     3241 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     3301 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     3361 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     3421 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     3481 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     3541 cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg
     3601 cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa
     3661 acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg
     3721 ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc
     3781 agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat
     3841 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     3901 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3961 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4021 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4081 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     4141 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4201 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4261 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4321 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4381 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4441 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4501 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4561 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4621 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4681 catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc cc
//
