LOCUS       sequence-12194-e        6874 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 6874)
  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 6874)
  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-gap16-ClopHensor
FEATURES             Location/Qualifiers
     source          1..6874
                     /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             955..978
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             join(985..987,988..1698)
                     /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(1027..1048)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1288..1307)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1635..1656
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1729..1746
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1759..2433
                     /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     2154..2173
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2468..2485)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2474..2698
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2744..3172
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2831..2850)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3041..3062
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3181..3201)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3186..3515
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3366..3501
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3428..3447
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3582..4376
                     /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(3636..3655)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4246..4265
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4550..4671
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4587..4606)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4641..4660
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4720..4736)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4720..4736)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4733..4755)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4744..4760
                     /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(4768..4774,4775..4792,4793..4798))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4813..4834
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4951..4968)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5122..5707)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5202..5221)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5878..6669,6670..6738))
                     /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     6501..6520
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6739..6843)
                     /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 gcatgaggag gaccaagcag gtggagaaga acgacgaggc tagctggagc
      961 cacccgcagt tcgaaaaagg cgccgtgagc aagggcgagg agctgttcac cggggtggtg
     1021 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     1081 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     1141 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     1201 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     1261 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     1321 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     1381 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     1441 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     1501 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     1561 gtgctgctgc ccgacaacca ctacctgagc taccagtccg ccctgagcaa agaccccaac
     1621 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     1681 atggacgagc tgtacaagcg cggatccgcg tctggtggtg gtggtggtct agttccacgt
     1741 ggatctgcct caggagcaga caacaccgag gacgtcatca aggagttcat gcagttcaag
     1801 gtgcgcatgg agggctccgt gaacggccac tacttcgaga tcgagggcga gggcgagggc
     1861 aagccctacg agggcaccca gaccgccaag ctgcaggtga ccaagggcgg ccccctgccc
     1921 ttcgcctggg acatcctgtc cccccagttc cagtacggct ccaaggccta cgtgaagcac
     1981 cccgccgaca tccccgacta catgaagctg tccttccccg agggcttcac ctgggagcgc
     2041 tccatgaact tcgaggacgg cggcgtggtg gaggtgcagc aggactcctc cctgcaggac
     2101 ggcaccttca tctacaaggt gaagttcaag ggcgtgaact tccccgccga cggccccgta
     2161 atgcagaaga agactgccgg ctgggagccc tccaccgaga agctgtaccc ccaggacggc
     2221 gtgctgaagg gcgagatctc ccacgccctg aagctgaagg acggcggcca ctacacctgc
     2281 gacttcaaga ccgtgtacaa ggccaagaag cccgtgcagc tgcccggcaa ccactacgtg
     2341 gactccaagc tggacatcac caaccacaac gaggactaca ccgtggtgga gcagtacgag
     2401 cacgccgagg cccgccactc cggctcccag tagtgctcta gagggcccgt ttaaacccgc
     2461 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2521 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2581 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2641 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2701 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2761 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2821 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2881 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2941 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3001 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3061 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3121 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     3181 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3241 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3301 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3361 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3421 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     3481 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     3541 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     3601 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     3661 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     3721 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     3781 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     3841 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     3901 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     3961 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     4021 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     4081 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     4141 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4201 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4261 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4321 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4381 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     4441 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4501 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     4561 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4621 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4681 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4741 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4801 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4861 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4921 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4981 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5041 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5101 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5161 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5221 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5281 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5341 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5401 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5461 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5521 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5581 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5641 aaccaccgct ggtagcggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5701 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5761 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5821 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5881 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5941 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6001 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6061 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6121 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6181 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6241 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6301 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6361 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6421 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6481 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6541 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6601 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6661 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6721 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6781 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6841 gcgcacattt ccccgaaaag tgccacctga cgtc
//
