LOCUS       sequence-396438-        7196 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Lime in synaptic vesicle lumen by fusion to synaptophysin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7196)
  AUTHORS   Shen Y, Wen Y, Sposini S, Vishwanath AA, Abdelfattah AS, Schreiter 
            ER, Lemieux MJ, de Juan-Sanz J, Perrais D, Campbell RE
  TITLE     Rational Engineering of an Improved Genetically Encoded pH Sensor 
            Based on Superecliptic pHluorin.
  JOURNAL   ACS Sens. 2023 Aug 25;8(8):3014-3022. doi: 
            10.1021/acssensors.3c00484. Epub 2023 Jul 23.
  PUBMED    37481776
REFERENCE   2  (bases 1 to 7196)
  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.3c00484"; journalName: "ACS Sens"; date: 
            "2023-08-25- 25"; volume: "8"; issue: "8"; pages: "3014-3022"
COMMENT     Sequence Label: CMV-synaptophysin-Lime
FEATURES             Location/Qualifiers
     source          1..7196
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             424..450
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             465..494
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             510..527
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(571..588)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    577..801
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      847..1275
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(934..953)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1144..1165
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1284..1304)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1289..1618
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1469..1604
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1531..1550
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1685..2479
                     /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(1739..1758)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2349..2368
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2653..2774
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2690..2709)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2744..2763
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2823..2839)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2823..2839)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2836..2858)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2847..2863
                     /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(2871..2877,2878..2895,2896..2901))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2916..2937
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3054..3071)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3225..3813)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3305..3324)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3984..4775,4776..4844))
                     /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     4607..4626
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4845..4949)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5024..5043)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5215..5594
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5595..5798
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5749..5769
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5843..5862
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5843..5861
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5916..5925
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 accggtggag ctcgaatcac atgcaaggaa ctgagggacc ctgtgacttc aggactcaac
       61 acctcggtgg tgtttggctt cctgaacctg gtgctctggg ttggcaacct atggttcgtg
      121 ttcaaggaga caggctgggc cgccccattc atgcgcgcac ctccaggcgc cccagaaaag
      181 caaccagctc ctggcgatgc ctacggcgat gcgggctatg ggcagggccc cggaggctat
      241 gggccccagg actcctacgg gcctcagggt ggttatcaac ccgattacgg gcagccagcc
      301 agcggcggtg gcggtggcta cgggcctcag ggcgactatg ggcagcaagg ctacggccaa
      361 cagggtgcgc ccacctcctt ctccaatcag atgtcggatc cgaagcttcg gtaccaagct
      421 gcctaccctt acgatgtgcc cgattacgcc tgatagctta cgtagaacaa aaactcatct
      481 cagaagagga tctgaatagc gccgtcgacc atcatcatca tcatcattga gtttaaacgg
      541 tctccagctt aagtttaaac cgctgatcag cctcgactgt gccttctagt tgccagccat
      601 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
      661 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
      721 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
      781 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
      841 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
      901 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
      961 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1021 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1081 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1141 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1201 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1261 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtcccaagg
     1321 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     1381 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     1441 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     1501 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     1561 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     1621 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     1681 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     1741 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     1801 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     1861 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     1921 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     1981 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     2041 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     2101 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     2161 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     2221 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     2281 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     2341 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     2401 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     2461 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     2521 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     2581 atcgttttcc gggacgccgg ctggatgatc ctccagcgca gggatctcat gctggagttc
     2641 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     2701 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     2761 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     2821 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     2881 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     2941 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     3001 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     3061 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3121 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3181 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3241 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3301 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3361 ctgccgctta ccggatacct gtccgccttt atcccttcgg gaagcgtggc gctttctcat
     3421 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3481 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3541 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3601 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3661 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3721 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3781 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3841 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3901 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3961 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4021 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4081 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4141 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4201 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4261 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4321 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4381 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4441 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4501 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4561 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4621 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4681 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4741 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4801 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4861 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4921 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     4981 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
     5041 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
     5101 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
     5161 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
     5221 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     5281 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     5341 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     5401 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     5461 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     5521 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     5581 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     5641 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     5701 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     5761 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
     5821 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
     5881 gtttaaacgg gccctctaga ctcgagctca agcttgccac catggacgtg gtgaatcagc
     5941 tggtggctgg gggtcagttc cgggtggtca aggagcccct tggcttcgtg aaggtgctgc
     6001 agtgggtctt tgccatcttc gcctttgcta cgtgcggcag ctacaccgga gagcttcggc
     6061 tgagcgtgga gtgtgccaac aagacggaga gtgccctcaa catcgaagtc gaatttgagt
     6121 acccattcag gctgcaccaa gtgtactttg atgcaccctc ctgcgttaaa gggggcacta
     6181 ccaagatctt cctagttggt gactactcct cctcggctga attctttgtc accgtggctg
     6241 tgtttgcctt cctctactcc atgggggccc tggccaccta catcttcctg cagaacaagt
     6301 accgagagaa caacaaaggg ccaatgatgg acttcctggc cacagcagtg ttcgctttca
     6361 tgtggctagt tagctcatcc gcctgggcca aaggcctgtc cgatgtgaag atggccactg
     6421 acccagagaa cattatcaag gagatgccta tgtgccgcca gacagatcta gccaccatga
     6481 tggtgagtaa aggagaagaa cttttcactg gagtggtgcc tatcctggtg gaactggatg
     6541 gcgacgtgaa cggccacaag ttcagcgtta gaggcgaagg cgaaggggat gccacaaacg
     6601 gcaagctgac cctgaagttc atcagcacca caggcaagct gcccgtgcct tggcctacac
     6661 tggtcacaac actgacctac ggcgtgcaga gcttcagcag ataccccgac cacatgaagc
     6721 ggcacgattt cttcaagagc gccatgccag agggctacgt gcaagagaga accatcagct
     6781 tcaaggacga cggcacctac aagaccagag ccgaagtgaa gttcgagggc gacaccctgg
     6841 tcaaccggat cgagctgaag ggcatcgact tcaaagagga cggcaacatc ctgggccaca
     6901 aacttgagta caacttcaac gaccaccagg tctacatcac cgccgacaag cagaagaacg
     6961 gcatcaaggc caacttcaag atccggcaca acgtggaaga tggcagcgtg cagctggccg
     7021 atcactacca gcagaacaca cccatcggag atggccctgt gctgctgccc gataaccact
     7081 acctgttcac caccagcaca ctgagcaagg accccaacga gaagcgggac cacatggtgc
     7141 tgctggaatt tgtgaccgcc gccggcatta cacatggcat ggatgaacta tacaaa
//
