LOCUS       sequence-275330-        6156 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses far-red fluorescent protein mNeptune2.5 in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6156)
  AUTHORS   Chu J, Haynes RD, Corbel SY, Li P, Gonzalez-Gonzalez E, Burg JS, 
            Ataie NJ, Lam AJ, Cranfill PJ, Baird MA, Davidson MW, Ng HL, Garcia 
            KC, Contag CH, Shen K, Blau HM, Lin MZ
  TITLE     Non-invasive intravital imaging of cellular differentiation with a 
            bright red-excitable fluorescent protein.
  JOURNAL   Nat Methods. 2014 Mar 16. doi: 10.1038/nmeth.2888.
  PUBMED    24633408
REFERENCE   2  (bases 1 to 6156)
  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: "Nat 
            Methods. 2014 Mar 16. doi: 10.1038/nmeth.2888."
COMMENT     Sequence Label: pcDNA3-mNeptune2.5
FEATURES             Location/Qualifiers
     source          1..6156
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             914..1648
                     /codon_start=1
                     /product="brighter version of the far-red fluorescent 
                     protein mNeptune2 with slightly blue-shifted spectra"
                     /label=mNeptune2.5
                     /translation="MVSKGEELIKENMHTKLYMEGTVNNHHFKCTHEGEGKPYEGTQTN
                     RIKVVEGGPLPFAFDILATCFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTVTQDTSLQDGCLIYNVKLRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRCD
                     MALKLVGGGHLHCNLKTTYRSKKPAKNLKMPGVYFVDRRLERIKEADNETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     promoter        complement(1709..1727)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1710..1727)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1747..1764)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1753..1977
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2023..2451
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2110..2129)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2320..2341
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2460..2480)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2465..2794
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2645..2780
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2707..2726
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2861..3655
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2915..2934)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3525..3544
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3829..3950
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3866..3885)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3920..3939
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3999..4015)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3999..4015)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4012..4034)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4023..4039
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4047..4053,4054..4071,4072..4077))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4092..4113
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4230..4247)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4401..4989)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4481..4500)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5160..5951,5952..6020))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5783..5802
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6021..6125)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccgatggtga gcaagggcga ggagctgatc aaggagaaca tgcacacgaa
      961 gctgtacatg gaaggcaccg tgaacaacca ccacttcaag tgcacccacg aaggggaggg
     1021 caagccctac gagggcaccc agaccaacag gattaaggtg gtggagggag gccccctgcc
     1081 gttcgcattc gacatcctgg ccacctgctt tatgtacggg agcaagacct tcatcaacca
     1141 cacccagggc atccccgatt tctttaagca gtccttccct gagggcttca catgggagag
     1201 agtcaccaca tacgaagacg ggggcgtgct taccgttacc caggacacca gcctccagga
     1261 cggctgcttg atctacaacg tcaagctcag aggggtgaac ttcccatcca acggccctgt
     1321 gatgcagaag aaaacactcg gctgggaggc cagtaccgag accctgtacc ccgctgacgg
     1381 cggcctggaa ggcagatgcg acatggccct gaagctcgtg ggcgggggcc acctgcactg
     1441 caacctgaag accacataca gatccaagaa acccgctaag aacctcaaga tgcccggcgt
     1501 ctactttgtg gaccgcagac tggaaagaat caaggaggcc gacaatgaga cctacgtcga
     1561 gcagcacgag gtggctgtgg ccagatactg cgacctccct agcaaactgg ggcacaaact
     1621 taatggcatg gacgagctgt acaagtaaga attctgcaga tatccatcac actggcggcc
     1681 gctcgagcat gcatctagag ggccctattc tatagtgtca cctaaatgct agagctcgct
     1741 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     1801 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     1861 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     1921 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     1981 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     2041 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2101 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2161 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2221 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2281 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2341 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2401 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     2461 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     2521 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     2581 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     2641 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     2701 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     2761 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
     2821 tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     2881 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     2941 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     3001 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     3061 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3121 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     3181 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3241 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3301 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     3361 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     3421 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     3481 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     3541 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     3601 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     3661 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     3721 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     3781 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     3841 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     3901 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     3961 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     4021 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4081 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4141 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4201 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4261 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4321 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4381 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4441 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4501 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4561 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4621 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4681 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4741 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4801 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4861 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4921 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4981 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5041 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5101 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5161 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5221 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5281 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5341 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5401 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5461 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5521 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5581 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5641 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5701 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5761 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5821 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5881 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5941 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6001 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6061 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6121 ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
