LOCUS       sequence-63670-e        6234 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 6234)
  AUTHORS   de la Fuente S, Fonteriz RI, de la Cruz PJ, Montero M, Alvarez J
  TITLE     Mitochondrial free [Ca(2+)] dynamics measured with a novel 
            low-Ca(2+) affinity aequorin probe.
  JOURNAL   Biochem J. 2012 Aug 1;445(3):371-6. doi: 10.1042/BJ20120423.
  PUBMED    22671130
REFERENCE   2  (bases 1 to 6234)
  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"; doi: "10"; journalName: 
            "Biochem J"; date: "2012-08-1- 1"; volume: "445"; issue: "3"; pages:
            "371-6"
COMMENT     Sequence Label: pcDNA3.1+/mit-2mutAEQ
FEATURES             Location/Qualifiers
     source          1..6234
                     /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             999..1073
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1104..1130
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     primer_bind     complement(1828..1845)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1834..2058
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2104..2532
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2191..2210)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2401..2422
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2541..2561)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2546..2875
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2726..2861
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2788..2807
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2942..3736
                     /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(2996..3015)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3606..3625
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3910..4031
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3947..3966)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4001..4020
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4080..4096)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4080..4096)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4093..4115)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4104..4120
                     /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(4128..4134,4135..4152,4153..4158))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4173..4194
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4311..4328)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4482..5067)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4562..4581)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5238..6029,6030..6098))
                     /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     5861..5880
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6099..6203)
                     /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 aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattcggc
      961 tacggctgac cgttttttgt ggtgtactcc gtgccatcat gtccgtcctg acgccgctgc
     1021 tgctgcgggg cttgacaggc tcggcccggc ggctcccagt gccgcgcgcc aagatccatt
     1081 cgttgccgcc ggaggggaag ctttatgatg ttcctgatta tgctagcctc aaacttacat
     1141 cagacttcga caacccaaga tggattggac gacacaagca tatgttcaat ttccttgatg
     1201 tcgaccacct tggaaaaatc tctcttgacg agatggtcta caaggcatct gatattgtca
     1261 tcaataacct tggagcaaca cctgagcaag ccaaacgaca caaagatgct gtagaagcct
     1321 tcttcggagg agctggaatg aaatatggtg tggaaactga ttggcctgca tatattgaag
     1381 gatggaaaaa attggctact gatgaattgg agaaatacgc caaaaacgaa ccaacgctca
     1441 tccgtatatg gggtgatgct ttgtttgata tcgtcgacaa agcccaaaat ggagccatta
     1501 cactggatga atggaaagca tacaccaaag ctgctggtat catccaatca tcagaagatt
     1561 gcgaggaaac attcagagtg tgcgatattg atgaaagtgg acaactcgat gttgatgaga
     1621 tgacaagaca acatttagga ttttggtaca ccatggatcc tgcttgcgaa aagctctacg
     1681 gtggagctgt cccctaagaa gctctacggt ggtgatgcac cctgggaaga tgatgtgatt
     1741 ttgaataaaa cactgatgaa ttctgcagat atccagcaca gtggcggccg ctcgagtcta
     1801 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     1861 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     1921 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     1981 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2041 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     2101 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2161 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2221 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2281 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2341 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2401 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2461 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2521 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     2581 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     2641 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2701 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     2761 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     2821 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     2881 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     2941 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3001 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3061 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3121 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3181 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3241 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3301 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3361 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     3421 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     3481 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     3541 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     3601 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     3661 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     3721 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     3781 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     3841 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     3901 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3961 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4021 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     4081 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4141 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4201 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4261 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4321 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4381 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4441 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4501 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4561 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4621 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4681 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4741 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4801 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4861 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4921 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4981 agctcttgat ccggcaaaca aaccaccgct ggtagcggtt tttttgtttg caagcagcag
     5041 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5101 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5161 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5221 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5281 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5341 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5401 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5461 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5521 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5581 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5641 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5701 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5761 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5821 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5881 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5941 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6001 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6061 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6121 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6181 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
