LOCUS       sequence-12159-e        6848 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 6848)
  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 6848)
  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-ClopHensor
FEATURES             Location/Qualifiers
     source          1..6848
                     /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             929..952
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             join(959..961,962..1672)
                     /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(1001..1022)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1262..1281)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1609..1630
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1703..1720
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1733..2407
                     /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     2128..2147
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2442..2459)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2448..2672
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2718..3146
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2805..2824)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3015..3036
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3155..3175)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3160..3489
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3340..3475
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3402..3421
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3556..4350
                     /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(3610..3629)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4220..4239
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4524..4645
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4561..4580)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4615..4634
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4694..4710)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4694..4710)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4707..4729)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4718..4734
                     /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(4742..4748,4749..4766,4767..4772))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4787..4808
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4925..4942)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5096..5681)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5176..5195)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5852..6643,6644..6712))
                     /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     6475..6494
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6713..6817)
                     /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 gtttaaactt aagcttggga tggctagctg gagccacccg cagttcgaaa aaggcgccgt
      961 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1021 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     1081 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1141 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     1201 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     1261 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1321 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1381 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     1441 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     1501 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1561 gagctaccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1621 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agcgcggatc
     1681 cgcgtctggt ggtggtggtg gtctagttcc acgtggatct gcctcaggag cagacaacac
     1741 cgaggacgtc atcaaggagt tcatgcagtt caaggtgcgc atggagggct ccgtgaacgg
     1801 ccactacttc gagatcgagg gcgagggcga gggcaagccc tacgagggca cccagaccgc
     1861 caagctgcag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccccca
     1921 gttccagtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacatgaa
     1981 gctgtccttc cccgagggct tcacctggga gcgctccatg aacttcgagg acggcggcgt
     2041 ggtggaggtg cagcaggact cctccctgca ggacggcacc ttcatctaca aggtgaagtt
     2101 caagggcgtg aacttccccg ccgacggccc cgtaatgcag aagaagactg ccggctggga
     2161 gccctccacc gagaagctgt acccccagga cggcgtgctg aagggcgaga tctcccacgc
     2221 cctgaagctg aaggacggcg gccactacac ctgcgacttc aagaccgtgt acaaggccaa
     2281 gaagcccgtg cagctgcccg gcaaccacta cgtggactcc aagctggaca tcaccaacca
     2341 caacgaggac tacaccgtgg tggagcagta cgagcacgcc gaggcccgcc actccggctc
     2401 ccagtagtgc tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     2461 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2521 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2581 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     2641 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     2701 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2761 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2821 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2881 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2941 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3001 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3061 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3121 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     3181 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3241 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3301 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     3361 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     3421 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3481 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     3541 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3601 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3661 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3721 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3781 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3841 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3901 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3961 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     4021 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     4081 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     4141 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     4201 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     4261 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     4321 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     4381 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     4441 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4501 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4561 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4621 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4681 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4741 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4801 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4861 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4921 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4981 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5041 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5101 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5161 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5221 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5281 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5341 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5401 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5461 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5521 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5581 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtttttttg
     5641 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5701 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5761 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5821 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5881 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5941 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6001 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6061 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6121 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6181 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6241 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6301 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6361 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6421 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6481 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6541 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6601 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6661 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6721 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6781 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6841 ctgacgtc
//
