LOCUS       sequence-424395-        5875 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 5875)
  AUTHORS   Ebert MS, Neilson JR, Sharp PA
  TITLE     MicroRNA sponges: competitive inhibitors of small RNAs in mammalian 
            cells.
  JOURNAL   Nat Methods. 2007 Sep . 4(9):721-6.
  PUBMED    17694064
REFERENCE   2  (bases 1 to 5875)
  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. 2007 Sep . 4(9):721-6."
COMMENT     Sequence Label: CMV-d2eGFP-empty
FEATURES             Location/Qualifiers
     source          1..5875
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(52..69)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    58..282
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(415..434)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    566..613
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             621..1643
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             621..1643
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    1773..1894
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1810..1829)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1864..1883
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1943..1959)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1943..1959)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1956..1978)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1967..1983
                     /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(1991..1997,1998..2015,2016..2021))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2174..2191)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2345..2933)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2425..2444)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3104..3895,3896..3964))
                     /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     3727..3746
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3965..4069)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4144..4163)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4335..4714
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4715..4918
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4869..4889
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4963..4982
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4963..4981
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5042..5051
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5048..5050,5051..5053,5054..5764,5765..5770,5771..5875,
                     1..18)
                     /codon_start=1
                     /product="EGFP destabilized by fusion to residues 422-461 
                     of mouse ornithine decarboxylase, giving an in vivo 
                     half-life of ~2 hours"
                     /label=d2EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKKLSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV
                     "
     primer_bind     complement(5093..5114)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5354..5373)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5701..5722
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 gctaggatca atgtgtaggc ggccgcctcg acgtttaaac ccgctgatca gcctcgactg
       61 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
      121 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
      181 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
      241 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
      301 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
      361 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
      421 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
      481 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
      541 attagggtga tggttcacgt acctagaagt tcctattccg aagttcctat tctctagaaa
      601 gtataggaac ttccttggcc aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc
      661 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc
      721 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
      781 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc
      841 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg
      901 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
      961 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
     1021 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg
     1081 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg
     1141 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg
     1201 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca
     1261 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
     1321 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
     1381 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct
     1441 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg
     1501 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg
     1561 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca
     1621 ctcgtccgag ggcaaaggaa tagcacgtac tacgagattt cgattccacc gccgccttct
     1681 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     1741 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     1801 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     1861 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     1921 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     1981 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     2041 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     2101 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     2161 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     2221 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     2281 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     2341 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     2401 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     2461 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     2521 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     2581 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     2641 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     2701 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     2761 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     2821 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     2881 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     2941 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3001 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3061 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3121 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     3181 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3241 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     3301 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     3361 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     3421 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     3481 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     3541 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     3601 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     3661 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     3721 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     3781 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     3841 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     3901 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     3961 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4021 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4081 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     4141 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     4201 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     4261 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     4321 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
     4381 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     4441 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     4501 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     4561 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     4621 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     4681 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
     4741 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
     4801 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
     4861 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
     4921 ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag
     4981 ggagacccaa gctggctagc gtttaaactt aagcttggta ccgagctcgg atccaccggt
     5041 cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     5101 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     5161 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     5221 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     5281 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     5341 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     5401 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     5461 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     5521 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     5581 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     5641 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     5701 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     5761 caagaagctt agccatggct tcccgccgga ggtggaggag caggatgatg gcacgctgcc
     5821 catgtcttgt gcccaggaga gcgggatgga ccgtcaccct gcagcctgtg cttct
//
