LOCUS       sequence-391714-        7356 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 7356)
  AUTHORS   Violin JD, Zhang J, Tsien RY, Newton AC
  TITLE     A genetically encoded fluorescent reporter reveals oscillatory 
            phosphorylation by protein kinase C.
  JOURNAL   J Cell Biol. 2003 Jun 9. 161(5):899-909.
  PUBMED    12782683
REFERENCE   2  (bases 1 to 7356)
  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: "J Cell 
            Biol. 2003 Jun 9. 161(5):899-909."
COMMENT     Sequence Label: CKAR-T/A
FEATURES             Location/Qualifiers
     source          1..7356
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      662..671
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(668..670,671..673,674..1384)
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             join(1943..1945,1946..2659)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     promoter        complement(2675..2693)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2676..2693)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     rep_origin      2989..3417
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3076..3095)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3286..3307
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3426..3446)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3431..3760
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3611..3746
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3673..3692
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3827..4621
                     /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(3881..3900)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4491..4510
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4795..4916
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4832..4851)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4886..4905
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4965..4981)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4965..4981)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4978..5000)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4989..5005
                     /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(5013..5019,5020..5037,5038..5043))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5058..5079
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5196..5213)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5367..5955)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5447..5466)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6126..6917,6918..6986))
                     /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     6749..6768
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6987..7091)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7166..7185)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg tccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
      721 gctggacggc gacgtaaacg gccacaggtt cagcgtgtcc ggcgagggcg agggcgatgc
      781 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
      841 gcccaccctc gtgaccaccc tgacctgggg cgtgcagtgc ttcagccgct accccgacca
      901 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgtac
      961 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     1021 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     1081 ggggcacaag ctggagtaca actacatcag ccacaacgtc tatatcaccg ccgacaagca
     1141 gaagaacggc atcaaggccc acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     1201 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     1261 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     1321 catggtcctg ctggagttcg tgaccgccgc cgggatcact cacggcatgg acgagctgta
     1381 caaggctggt accaagggta atggtaggtt tttaacttta aaaccattgc ctgacagcat
     1441 tattcaagaa agcctggaga ttcagcaagg tgtgaatcca tttttcattg gtagatccga
     1501 ggattgcaat tgtaaaattg aagacagtag gttgtctcga gttcattgct tcattttcaa
     1561 aaagaggcat gctgtaggca aaagcatgta tgaatctccg gcacaaggtt tagatgatat
     1621 ttggtattgc cacaccggaa ctaacgtgag ctatttaaat aataaccgca tgatacaggg
     1681 tacgaaattc cttttacaag acggagatga aatcaagatc atttgggata aaaacaataa
     1741 atttgtcatt ggctttaaag tggaaattaa cgatactaca ggtctgttta acgagggatt
     1801 aggtatgtta caagaacaaa gagtagtact taagcaaaca gccgaagaaa aagatttggt
     1861 gaaaaagtta ggaggatccg gaggtcgatt cagaagattc caggcgttga agatcaaggc
     1921 taaggcaggt ggatctggtg gagtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1981 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     2041 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     2101 gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcctgaagt gcttcgcccg
     2161 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     2221 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     2281 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     2341 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     2401 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     2461 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     2521 gctgctgccc gacaaccact acctgagcta ccagtccgcc ctgagcaaag accccaacga
     2581 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctcacggcat
     2641 ggacgagctg tacaagtaat ctagagggcc ctattctata gtgtcaccta aatgctagag
     2701 ctcgctgatc agcctcgact atgccttcta gttgccagcc atctgttgtt tgcccctccc
     2761 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2821 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2881 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2941 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     3001 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3061 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3121 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3181 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3241 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3301 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3361 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     3421 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3481 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3541 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3601 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3661 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     3721 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     3781 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3841 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3901 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3961 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     4021 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     4081 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4141 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     4201 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     4261 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4321 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     4381 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4441 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4501 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4561 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4621 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4681 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4741 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4801 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4861 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4921 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4981 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     5041 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     5101 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5161 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5221 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5281 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5341 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5401 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5461 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5521 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5581 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5641 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5701 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5761 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5821 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5881 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5941 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6001 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6061 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6121 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6181 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6241 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6301 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6361 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6421 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6481 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6541 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6601 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6661 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6721 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6781 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6841 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6901 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6961 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7021 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7081 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
     7141 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     7201 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
     7261 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
     7321 cgctgcttcg cgatgtacgg gccagatata cgcgtt
//
