LOCUS       sequence-12160-e        6958 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6958)
  AUTHORS   Arosio D, Ricci F, Marchetti L, Gualdani R, Albertazzi L, Beltram F
  TITLE     Simultaneous intracellular chloride and pH measurements using a 
            GFP-based sensor.
  JOURNAL   Nat Methods. 2010 Jul . 7(7):516-8.
  PUBMED    20581829
REFERENCE   2  (bases 1 to 6958)
  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. 2010 Jul . 7(7):516-8."
COMMENT     Sequence Label: pcDNA3-NPY-ClopHensor
FEATURES             Location/Qualifiers
     source          1..6958
                     /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             1039..1062
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             join(1069..1071,1072..1782)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1111..1132)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1372..1391)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1719..1740
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1813..1830
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1843..2517
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Strongin et al., 2007)"
                     /label=DsRed-Monomer
                     /note="mammalian codon-optimized"
                     /translation="DNTEDVIKEFMQFKVRMEGSVNGHYFEIEGEGEGKPYEGTQTAKL
                     QVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYMKLSFPEGFTWERSMNFEDGGVV
                     EVQQDSSLQDGTFIYKVKFKGVNFPADGPVMQKKTAGWEPSTEKLYPQDGVLKGEISHA
                     LKLKDGGHYTCDFKTVYKAKKPVQLPGNHYVDSKLDITNHNEDYTVVEQYEHAEARHSG
                     SQ"
     primer_bind     2238..2257
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2552..2569)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2558..2782
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2828..3256
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2915..2934)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3125..3146
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3265..3285)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3270..3599
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3450..3585
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3512..3531
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3666..4460
                     /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(3720..3739)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4330..4349
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4634..4755
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4671..4690)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4725..4744
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4804..4820)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4804..4820)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4817..4839)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4828..4844
                     /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(4852..4858,4859..4876,4877..4882))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4897..4918
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5035..5052)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5206..5791)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5286..5305)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5962..6753,6754..6822))
                     /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     6585..6604
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6823..6927)
                     /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 atgctaggta acaagcgact ggggctgtcc ggactgaccc tcgccctgtc cctgctcgtg
      961 tgcctgggtg cgctggccga ggcgtacccc tccaagccgg acaacccggg cgaggacgca
     1021 ccagcggagg acgctagctg gagccacccg cagttcgaaa aaggcgccgt gagcaagggc
     1081 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1141 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1201 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1261 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1321 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1381 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1441 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1501 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     1561 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1621 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagctaccag
     1681 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1741 accgccgccg ggatcactct cggcatggac gagctgtaca agcgcggatc cgcgtctggt
     1801 ggtggtggtg gtctagttcc acgtggatct gcctcaggag cagacaacac cgaggacgtc
     1861 atcaaggagt tcatgcagtt caaggtgcgc atggagggct ccgtgaacgg ccactacttc
     1921 gagatcgagg gcgagggcga gggcaagccc tacgagggca cccagaccgc caagctgcag
     1981 gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccccca gttccagtac
     2041 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacatgaa gctgtccttc
     2101 cccgagggct tcacctggga gcgctccatg aacttcgagg acggcggcgt ggtggaggtg
     2161 cagcaggact cctccctgca ggacggcacc ttcatctaca aggtgaagtt caagggcgtg
     2221 aacttccccg ccgacggccc cgtaatgcag aagaagactg ccggctggga gccctccacc
     2281 gagaagctgt acccccagga cggcgtgctg aagggcgaga tctcccacgc cctgaagctg
     2341 aaggacggcg gccactacac ctgcgacttc aagaccgtgt acaaggccaa gaagcccgtg
     2401 cagctgcccg gcaaccacta cgtggactcc aagctggaca tcaccaacca caacgaggac
     2461 tacaccgtgg tggagcagta cgagcacgcc gaggcccgcc actccggctc ccagtagtgc
     2521 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     2581 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     2641 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     2701 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     2761 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     2821 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2881 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2941 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     3001 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     3061 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3121 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     3181 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     3241 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     3301 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     3361 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3421 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3481 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     3541 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     3601 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     3661 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     3721 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     3781 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     3841 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     3901 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     3961 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     4021 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     4081 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     4141 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     4201 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     4261 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     4321 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     4381 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     4441 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     4501 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     4561 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     4621 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4681 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     4741 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     4801 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4861 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4921 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4981 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5041 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5101 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5161 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5221 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5281 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5341 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5401 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5461 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5521 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5581 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5641 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5701 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtttttttg tttgcaagca
     5761 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5821 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5881 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5941 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6001 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6061 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6121 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6181 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6241 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6301 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6361 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6421 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6481 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6541 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6601 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6661 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6721 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6781 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6841 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6901 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
