LOCUS       sequence-237605-        7478 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 7478)
  AUTHORS   Palmer AE, Giacomello M, Kortemme T, Hires SA, Lev-Ram V, Baker D, 
            Tsien RY
  TITLE     Ca2+ indicators based on computationally redesigned 
            calmodulin-peptide pairs.
  JOURNAL   Chem Biol. 2006 May;13(5):521-30.
  PUBMED    16720273
REFERENCE   2  (bases 1 to 7478)
  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: "Chem 
            Biol."; date: "2006-05"; volume: "13(5)"; pages: "521-30"
COMMENT     Sequence Label: pcDNA-D3cpv
FEATURES             Location/Qualifiers
     source          1..7478
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        67..446
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        447..650
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     601..621
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     695..714
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        695..713
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      733..742
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             754..771
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             775..807
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(811..819,820..834)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(841..843,844..846,847..1359)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIE"
     CDS             2065..2799
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRD
                     HMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     promoter        complement(2863..2881)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2864..2881)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2901..2918)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2907..3131
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3177..3605
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3264..3283)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3474..3495
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3614..3634)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3619..3948
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3799..3934
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3861..3880
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4015..4809
                     /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(4069..4088)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4679..4698
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4983..5104
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5020..5039)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5074..5093
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5153..5169)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5153..5169)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5166..5188)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5177..5193
                     /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(5201..5207,5208..5225,5226..5231))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5246..5267
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5384..5401)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5555..6143)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5635..5654)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6314..7105,7106..7174))
                     /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     6937..6956
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7175..7279)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7354..7373)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
       61 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      121 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      181 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      241 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      301 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      361 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      421 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      481 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      541 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      601 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
      661 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
      721 aagcttgcgg ccgccaccat ggtgcggggt tctcatcatc atcatcatca tggtatggct
      781 agcatgactg gtggacagca aatgggtcgg gatctgtacg acgatgacga taaggatccc
      841 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      901 ggcgacgtaa acggccacag gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      961 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1021 ctcgtgacca ccctgacctg gggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1081 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg taccatcttc
     1141 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1201 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1261 aagctggagt acaactacat cagccacaac gtctatatca ccgccgacaa gcagaagaac
     1321 ggcatcaagg cccacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1381 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1441 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1501 ctgctggagt tcgtgaccgc cgcccgcatg catgaccaac tgacagaaga gcagattgca
     1561 gagttcaaag aagccttctc attattggac aaggatgggg acggcaccat caccacaaag
     1621 gaacttggca ccgctctgag gtcgcttgga caaaacccaa cggaagcaga attgcaggat
     1681 atgatcaatg aagtcgatgc tgatggcaat ggaacgattt actttcctga atttcttact
     1741 atgatggcta gaaaaatgaa ggacacagac agcgaagagg aaatccgaga agcattccgt
     1801 gtttttgaca aggatgggaa cggctacatc agcgctgctg aattacgtca cgtcatgaca
     1861 aacctcgggg agaagttaac agatgaagaa gttgatgaaa tgataaggga agcagatatc
     1921 gatggtgatg gccaagtaaa ctatgaagag tttgtacaaa tgatgacagc aaaggggggg
     1981 aagaggcgct ggcagaaaac aggccatgct gtccgagcat tcggaagact gaagaagatc
     2041 tccagctccg gggcactgga gctcatggac ggcggcgtgc agctcgccga ccactaccag
     2101 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac
     2161 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2221 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggtgg cagcggtggc
     2281 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     2341 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2401 ggcaagctga ccctgaagct gatctgcacc accggcaagc tgcccgtgcc ctggcccacc
     2461 ctcgtgacca ccctgggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag
     2521 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2581 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2641 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2701 aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa gcagaagaac
     2761 ggcatcaagg ccaacttcaa gatccgccac aacatcgagt aagaattctg cagatatcca
     2821 tcacactggc ggccgctcga gcatgcatct agagggccct attctatagt gtcacctaaa
     2881 tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     2941 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3001 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3061 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3121 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     3181 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3241 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3301 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3361 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3421 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3481 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3541 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3601 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     3661 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     3721 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3781 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3841 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     3901 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     3961 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     4021 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4081 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     4141 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     4201 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4261 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     4321 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     4381 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     4441 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     4501 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     4561 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     4621 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     4681 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4741 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     4801 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     4861 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4921 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4981 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5041 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5101 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5161 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5221 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5281 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5341 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5401 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5461 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5521 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5581 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5641 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5701 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5761 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5821 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5881 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5941 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6001 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6061 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6121 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6181 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6241 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6301 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6361 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6421 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6481 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6541 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6601 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6661 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6721 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6781 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6841 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6901 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6961 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7021 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7081 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7141 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7201 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7261 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     7321 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     7381 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     7441 ttaagctaca acaaggcaag gcttgaccga caattgca
//
