LOCUS       sequence-12161-e        6886 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 6886)
  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 6886)
  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-PalmPalm-ClopHensor
FEATURES             Location/Qualifiers
     source          1..6886
                     /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             967..990
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             join(997..999,1000..1710)
                     /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(1039..1060)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1300..1319)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1647..1668
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1741..1758
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1771..2445
                     /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     2166..2185
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2480..2497)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2486..2710
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2756..3184
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2843..2862)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3053..3074
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3193..3213)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3198..3527
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3378..3513
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3440..3459
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3594..4388
                     /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(3648..3667)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4258..4277
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4562..4683
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4599..4618)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4653..4672
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4732..4748)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4732..4748)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4745..4767)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4756..4772
                     /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(4780..4786,4787..4804,4805..4810))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4825..4846
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4963..4980)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5134..5719)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5214..5233)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5890..6681,6682..6750))
                     /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     6513..6532
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6751..6855)
                     /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 atgctgtgct gtatgagaag aaccaaacag gttgaaaaga atgatgagga ccaaaagatc
      961 gctagctgga gccacccgca gttcgaaaaa ggcgccgtga gcaagggcga ggagctgttc
     1021 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1081 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     1141 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     1201 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     1261 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     1321 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1381 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     1441 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     1501 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     1561 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gctaccagtc cgccctgagc
     1621 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1681 atcactctcg gcatggacga gctgtacaag cgcggatccg cgtctggtgg tggtggtggt
     1741 ctagttccac gtggatctgc ctcaggagca gacaacaccg aggacgtcat caaggagttc
     1801 atgcagttca aggtgcgcat ggagggctcc gtgaacggcc actacttcga gatcgagggc
     1861 gagggcgagg gcaagcccta cgagggcacc cagaccgcca agctgcaggt gaccaagggc
     1921 ggccccctgc ccttcgcctg ggacatcctg tccccccagt tccagtacgg ctccaaggcc
     1981 tacgtgaagc accccgccga catccccgac tacatgaagc tgtccttccc cgagggcttc
     2041 acctgggagc gctccatgaa cttcgaggac ggcggcgtgg tggaggtgca gcaggactcc
     2101 tccctgcagg acggcacctt catctacaag gtgaagttca agggcgtgaa cttccccgcc
     2161 gacggccccg taatgcagaa gaagactgcc ggctgggagc cctccaccga gaagctgtac
     2221 ccccaggacg gcgtgctgaa gggcgagatc tcccacgccc tgaagctgaa ggacggcggc
     2281 cactacacct gcgacttcaa gaccgtgtac aaggccaaga agcccgtgca gctgcccggc
     2341 aaccactacg tggactccaa gctggacatc accaaccaca acgaggacta caccgtggtg
     2401 gagcagtacg agcacgccga ggcccgccac tccggctccc agtagtgctc tagagggccc
     2461 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2521 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2581 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2641 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     2701 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     2761 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2821 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2881 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2941 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3001 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3061 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3121 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3181 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3241 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3301 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3361 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3421 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     3481 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     3541 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     3601 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3661 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3721 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     3781 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     3841 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3901 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     3961 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4021 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4081 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     4141 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4201 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4261 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4321 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4381 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4441 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4501 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4561 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4621 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4681 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     4741 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4801 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4861 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4921 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4981 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5041 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5101 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5161 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5221 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5281 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5341 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5401 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5461 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5521 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5581 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5641 atccggcaaa caaaccaccg ctggtagcgg tttttttgtt tgcaagcagc agattacgcg
     5701 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5761 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5821 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5881 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5941 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6001 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6061 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6121 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6181 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6241 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6301 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6361 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6421 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6481 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6541 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6601 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6661 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6721 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6781 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6841 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
