LOCUS       sequence-320109-        4240 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for COMET promoters, expresses EYFP reporter upon promoter 
            activation. BsaI sites produce non-palindromic sticky ends..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4240)
  AUTHORS   Donahue PS, Draut JW, Muldoon JJ, Edelstein HI, Bagheri N, Leonard 
            JN
  TITLE     The COMET toolkit for composing customizable genetic programs in 
            mammalian cells.
  JOURNAL   Nat Commun. 2020 Feb 7;11(1):779. doi: 10.1038/s41467-019-14147-5.
  PUBMED    32034124
REFERENCE   2  (bases 1 to 4240)
  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 
            Commun."; date: "2020-02-7"; volume: "11(1):779. doi"; pages: " 
            10.1038/s41467-019-14147-5"
COMMENT     Sequence Label: pPD540 Reporter Template v3
FEATURES             Location/Qualifiers
     source          1..4240
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      115..124
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(121..123,124..126,127..840)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(166..187)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(427..446)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     774..795
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(885..902)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    891..1115
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1161..1589
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1248..1267)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1458..1479
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1612..1808
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1659..1794
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1721..1740
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    1928..2049
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1965..1984)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2019..2038
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2098..2114)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2098..2114)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2111..2133)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2122..2138
                     /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(2146..2152,2153..2170,2171..2176))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2191..2212
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2329..2346)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2500..3088)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2580..2599)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3259..4050,4051..4119))
                     /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     3882..3901
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4120..4224)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtgccacctg acgtcgacgg atcgggagat ctgcgagcga gaccacaggt ctcgacgctc
       61 tctagagggt atataatggg ggccactagt ctactaccag agctcatcgc tagcgccacc
      121 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      181 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      241 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      301 ctcgtgacca ccttcggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag
      361 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      421 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      481 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      541 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
      601 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      661 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      721 tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      781 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtag
      841 gcggccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
      901 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
      961 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     1021 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagtcaa
     1081 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     1141 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     1201 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     1261 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     1321 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     1381 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     1441 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     1501 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     1561 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttaggctca
     1621 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     1681 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     1741 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     1801 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg aggttgggct tcggaatcgt
     1861 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     1921 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     1981 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2041 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     2101 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     2161 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     2221 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     2281 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     2341 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     2401 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     2461 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     2521 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     2581 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     2641 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     2701 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2761 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2821 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     2881 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     2941 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3001 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3061 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3121 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     3181 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3241 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3301 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3361 gggcttacca tctggcccca gtgctgcaat gataccgcga gatccacgct caccggctcc
     3421 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3481 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3541 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3601 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3661 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3721 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     3781 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     3841 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     3901 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     3961 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4021 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4081 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     4141 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4201 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
//
