LOCUS       sequence-470683-        7248 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing NPY tag with pH sensor, pHCitron (pH-Lemon with 
            mCitrine), in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7248)
  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 7248)
  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 pHCitron
FEATURES             Location/Qualifiers
     source          1..7248
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(220..1014)
                     /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(331..350)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     941..960
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1081..1410)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1095..1230
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1149..1168)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     1395..1415
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(1424..1852)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1534..1555)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1746..1765
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1898..2122
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2111..2128
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(2278..2988,2989..2991,2992..2994))
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2320..2341)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4085..4104)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4086..4104)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4149..4352)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4178..4198)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4353..4732
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4904..4923
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4998..5102
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5103..5171,5172..5963)
                     /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     complement(5321..5340)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6134..6722
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6623..6642
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6876..6893
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7010..7031
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7046..7051,7052..7069,7070..7076)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7084..7100
                     /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)."
     primer_bind     7089..7111
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7108..7124
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7108..7124
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    join(7173..7248,1..46)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7184..7203)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     join(7238..7248,1..9)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttgggg tgggcgaaga
       61 actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg aaaacgattc
      121 cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg
      181 gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact cgtcaagaag
      241 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
      301 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
      361 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
      421 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
      481 catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
      541 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
      601 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
      661 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
      721 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
      781 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cctgcagttc
      841 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
      901 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
      961 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     1021 cgatcctcat cctgtctctt gatcagatcc gaaaatggat atacaagctc ccgggagctt
     1081 tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc
     1141 agaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg gcggagaatg
     1201 ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc
     1261 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     1321 acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
     1381 tggggacttt ccacacccta actgacacac attccacaga attaattcgc gttaaatttt
     1441 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     1501 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     1561 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     1621 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     1681 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     1741 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     1801 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtggggatac
     1861 cccctagagc cccagctggt tctttccgcc tcagaagcca tagagcccac cgcatcccca
     1921 gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt cctgccccac cccacccccc
     1981 agaatagaat gacacctact cagacaatgc gatgcaattt cctcatttta ttaggaaagg
     2041 acagtgggag tggcaccttc cagggtcaag gaaggcacgg gggaggggca aacaacagat
     2101 ggctggcaac tagaaggcac agtcgaggct gatcagcggg tttaaacggg ccctctagac
     2161 tcgagcggcc gcttacagac aagctaagct gaagaggctg aggagcagga ctgagggcag
     2221 gctgaagcgc agggcccctg ccgcgttgca gtcggtaccc taagactctt aggtaccctt
     2281 gtacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca gcaggaccat
     2341 gtgatcgcgc ttctcgttgg ggtctttgct cagcttggac tggtagctca ggtagtggtt
     2401 gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag
     2461 ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga tgccgttctt
     2521 ctgcttgtcg gccatgatat agacgttgtg gctgttgtag ttgtactcca gcttgtgccc
     2581 caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt
     2641 gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat
     2701 ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat
     2761 gtggtcgggg tagcgggcga agcacatcag gccgtagccg aaggtggtca cgagggtggg
     2821 ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt
     2881 ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag
     2941 ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca ccatgaattc
     3001 cgaaccacca ccgcctttgt acagttcatc catgcccagg gtaatgcccg cggctgtaac
     3061 aaattccaaa agaaccatgt gatctctttt ctcattcgga tctttcgaca gtttgctctg
     3121 ggtggacagg tagtggttat ccggcagcag cacagggccg tcaccaatcg gggtgttctg
     3181 ctggtaatga tccgccagtt gcaccccacc gtcctcaata ttatgacgga tcttaaaatt
     3241 tgctttgatg ccgtttttct gtttatcagc ggtgatatac acgttatcgc taaaataatt
     3301 atattccagt ttatgaccca gaatgtttcc atcctcctta aaatcaatgc ccttcagttc
     3361 aatgcggttg acgagggtgt cgccctcgaa tttcacttca gcacgggtct tgtagttgcc
     3421 gtcgtctttg aaaaagatgg tgcgctcttg aacgtagcct tccggcattg cgctcttgaa
     3481 gaaatcatgt tgcttcatgt ggtccggata acgggcgaag cattgaacac cccaagacaa
     3541 agtcgttacc agtgtaggcc acgggaccgg taacttaccg gtggtgcaga taaacttcag
     3601 ggtcagtttg ccgtacgtcg cgtcaccctc accctcaccg gaaacgctga acttgtggcc
     3661 gttcacgtcg ccgtccaact cgaccagaat cggaaccacg ccggtgaaca actcttctcc
     3721 ttttgaaact actgggggat cccgggcccg gggcaccgtc cacattgcag ggtcttcaag
     3781 ccgagttctg ggaacatttt ctgtgctttc tctcatcaag aggtctgaaa tcagtgtctc
     3841 tgggctggat cgttttccat atctctgcct ggtgatgagg ttgatgtagt gtcgcagcgc
     3901 cgagtagtat ctggccatgt cctccgctgg tgcgtcctcg cccgggttgt ccggcttgga
     3961 ggggtacgcc tcggccagcg cacccaggca cacgagcagg gacagggcga gggtcagtcc
     4021 ggacagcccc agtcgcttgt tacctagcat aagcttaagt ttaaacgcta gccagcttgg
     4081 gtctccctat agtgagtcgt attaatttcg ataagccagt aagcagtggg ttctctagtt
     4141 agccagagag ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc
     4201 aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa
     4261 ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca
     4321 cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag
     4381 atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc
     4441 catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact
     4501 gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt
     4561 ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt
     4621 cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga
     4681 actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg tcaacgcgta
     4741 tatctggccc gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag attcttcatg
     4801 caattgtcgg tcaagccttg ccttgttgta gcttaaattt tgctcgcgca ctactcagcg
     4861 acctccaaca cacaagcagg gagcagatac tggcttaact atgcggcatc agagcagatt
     4921 gtactgagag tgcaccatag gggatcggga gatctcccga tccgtcgacg tcaggtggca
     4981 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5041 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5101 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5161 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5221 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5281 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5341 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5401 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5461 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5521 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5581 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5641 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     5701 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     5761 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     5821 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     5881 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5941 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6001 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6061 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6121 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6181 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6241 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6301 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6361 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6421 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6481 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6541 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6601 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6661 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6721 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6781 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     6841 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     6901 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     6961 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     7021 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     7081 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     7141 tctagctaga ggtcgacggt atacagacat gataagatac attgatgagt ttggacaaac
     7201 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtga
//
