LOCUS       sequence-470434-        8576 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing NPY tag with pH sensor, pHLeon (pH-Lemon with 
            mNeonGreen), in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8576)
  AUTHORS   Lin W, Tseng K, Fraser SE, Junge J, White KL
  TITLE     Decoding Insulin Secretory Granule Maturation Using Genetically 
            Encoded pH Sensors.
  JOURNAL   ACS Sens. 2024 Nov 22;9(11):6032-6039. doi: 
            10.1021/acssensors.4c01885. Epub 2024 Nov 6.
  PUBMED    39504473
REFERENCE   2  (bases 1 to 8576)
  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: 
            "10.1021/acssensors.4c01885"; journalName: "ACS Sens"; date: 
            "2024-11-22- 22"; volume: "9"; issue: "11"; pages: "6032-6039"
COMMENT     Sequence Label: pcDNA3.1 NPY pHLeon
FEATURES             Location/Qualifiers
     source          1..8576
                     /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"
     primer_bind     complement(1558..1577)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1990..2697
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
                     LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDG
                     ASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIST
                     FKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK
                     AFTDVMGMDELYK"
     regulatory      2242..2251
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2829..2846)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2835..3059
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3105..3533
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3192..3211)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3402..3423
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3542..3562)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3547..3876
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3727..3862
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3789..3808
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3943..4737
                     /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(3997..4016)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4607..4626
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4911..5032
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4948..4967)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5002..5021
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5081..5097)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5081..5097)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5094..5116)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5105..5121
                     /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(5129..5135,5136..5153,5154..5159))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5174..5195
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5312..5329)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5483..6071)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5563..5582)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7583..8371,8372..8440))
                     /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
                     LIKH"
     primer_bind     8203..8222
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8441..8545)
                     /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 gctggctagc
      901 gtttaaacgg gccctctaga ctcgagcggc cgcatgctag gtaacaagcg actggggctg
      961 tccggactga ccctcgccct gtccctgctc gtgtgcctgg gtgcgctggc cgaggcgtac
     1021 ccctccaagc cggacaaccc gggcgaggac gcaccagcgg aggacatggc cagatactac
     1081 tcggcgctgc gacactacat caacctcatc accaggcaga gatatggaaa acgatccagc
     1141 ccagagacac tgatttcaga cctcttgatg agagaaagca cagaaaatgt tcccagaact
     1201 cggcttgaag accctgcaat gtggacggtg ccccgggccc gggatccccc agtagtttca
     1261 aaaggagaag agttgttcac cggcgtggtt ccgattctgg tcgagttgga cggcgacgtg
     1321 aacggccaca agttcagcgt ttccggtgag ggtgagggtg acgcgacgta cggcaaactg
     1381 accctgaagt ttatctgcac caccggtaag ttaccggtcc cgtggcctac actggtaacg
     1441 actttgtctt ggggtgttca atgcttcgcc cgttatccgg accacatgaa gcaacatgat
     1501 ttcttcaaga gcgcaatgcc ggaaggctac gttcaagagc gcaccatctt tttcaaagac
     1561 gacggcaact acaagacccg tgctgaagtg aaattcgagg gcgacaccct cgtcaaccgc
     1621 attgaactga agggcattga ttttaaggag gatggaaaca ttctgggtca taaactggaa
     1681 tataattatt ttagcgataa cgtgtatatc accgctgata aacagaaaaa cggcatcaaa
     1741 gcaaatttta agatccgtca taatattgag gacggtgggg tgcaactggc ggatcattac
     1801 cagcagaaca ccccgattgg tgacggccct gtgctgctgc cggataacca ctacctgtcc
     1861 acccagagca aactgtcgaa agatccgaat gagaaaagag atcacatggt tcttttggaa
     1921 tttgttacag ccgcgggcat taccctgggc atggatgaac tgtacaaagg cggtggtggt
     1981 tcggaattcg tgagcaaggg cgaggaggat aacatggcct ctctcccagc gacacatgag
     2041 ttacacatct ttggctccat caacggtgtg gactttgaca tggtgggtca gggcaccggc
     2101 aatccaaatg atggttatga ggagttaaac ctgaagtcca ccaagggtga cctccagttc
     2161 tccccctgga ttctggtccc tcatatcggg tatggcttcc atcagtacct gccctaccct
     2221 gacgggatgt cgcctttcca ggccgccatg gtagatggct ccggctacca agtccatcgc
     2281 acaatgcagt ttgaagatgg tgcctccctt actgttaact accgctacac ctacgaggga
     2341 agccacatca aaggagaggc ccaggtgaag gggactggtt tccctgctga cggtcctgtg
     2401 atgaccaact cgctgaccgc tgcggactgg tgcaggtcga agaagactta ccccaacgac
     2461 aaaaccatca tcagtacctt taagtggagt tacaccactg gaaatggcaa gcgctaccgg
     2521 agcactgcgc ggaccaccta cacctttgcc aagccaatgg cggctaacta tctgaagaac
     2581 cagccgatgt acgtgttccg taagacggag ctcaagcact ccaagaccga gctcaacttc
     2641 aaggagtggc aaaaggcctt taccgatgtg atgggcatgg acgagctgta caagtaaggt
     2701 acctaagagt cttagggtac cgactgcaac gcggcagggg ccctgcgctt cagcctgccc
     2761 tcagtcctgc tcctcagcct cttcagctta gcttgtctgt aaaagcttaa gtttaaaccg
     2821 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2881 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2941 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     3001 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     3061 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     3121 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3181 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3241 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3301 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3361 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3421 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3481 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     3541 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3601 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3661 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3721 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3781 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     3841 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     3901 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     3961 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     4021 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     4081 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     4141 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     4201 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     4261 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     4321 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     4381 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     4441 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     4501 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     4561 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     4621 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     4681 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     4741 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     4801 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4861 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     4921 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4981 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     5041 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     5101 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     5161 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     5221 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5281 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5341 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5401 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5461 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5521 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5581 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5641 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5701 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5761 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5821 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5881 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5941 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6001 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6061 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6121 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6181 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6241 gttaccaatg cttaatcctg atgaatcccc taatgatttt ggtaaaaatc attaagttaa
     6301 ggtggataca catcttgtca tatgatcaaa tggtttcgcg aaaaatcaat aatcagacaa
     6361 caagatgtgc gaactcgata ttttacacga ctctctttac caattctgcc ccgaattaca
     6421 cttaaaacga ctcaacagct taacgttggc ttgccacgca ttacttgact gtaaaactct
     6481 cactcttacc gaacttggcc gtaacctgcc aaccaaagcg agaacaaaac ataacatcaa
     6541 acgaatcgac cgattgttag gtaatcgtca cctccacaaa gagcgactcg ctgtataccg
     6601 ttggcatgct agctttatct gttcgggcaa tacgatgccc attgtacttg ttgactggtc
     6661 tgatattcgt gagcaaaaac gacttatggt attgcgagct tcagtcgcac tacacggtcg
     6721 ttctgttact ctttatgaga aagcgttccc gctttcagag caatgttcaa agaaagctca
     6781 tgaccaattt ctagccgacc ttgcgagcat tctaccgagt aacaccacac cgctcattgt
     6841 cagtgatgct ggctttaaag tgccatggta taaatccgtt gagaagctgg gttggtactg
     6901 gttaagtcga gtaagaggaa aagtacaata tgcagaccta ggagcggaaa actggaaacc
     6961 tatcagcaac ttacatgata tgtcatctag tcactcaaag actttaggct ataagaggct
     7021 gactaaaagc aatccaatct catgccaaat tctattgtat aaatctcgct ctaaaggccg
     7081 aaaaaatcag cgctcgacac ggactcattg tcaccacccg tcacctaaaa tctactcagc
     7141 gtcggcaaag gagccatggg ttctagcaac taacttacct gttgaaattc gaacacccaa
     7201 acaacttgtt aatatctatt cgaagcgaat gcagattgaa gaaaccttcc gagacttgaa
     7261 aagtcctgcc tacggactag gcctacgcca tagccgaacg agcagctcag agcgttttga
     7321 tatcatgctg ctaatcgccc tgatgcttca actaacatgt tggcttgcgg gcgttcatgc
     7381 tcagaaacaa ggttgggaca agcacttcca ggctaacaca gtcagaaatc gaaacgtact
     7441 ctcaacagtt cgcttaggca tggaagtttt gcggcattct ggctacacaa taacaaggga
     7501 agacttactc gtggctgcaa ccctactagc tcaaaattta ttcacacatg gttacgcttt
     7561 ggggaaatta tgaggggatc tctcagtgct taatcagtga ggcacctatc tcagcgatct
     7621 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     7681 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     7741 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     7801 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     7861 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     7921 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     7981 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     8041 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     8101 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     8161 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     8221 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     8281 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     8341 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     8401 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     8461 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     8521 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
