LOCUS       sequence-323526-        4421 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EYFP reporter vector for the ZF43x4-Spaced-Mid promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4421)
  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 4421)
  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: pPD135 ZF43x4-Spaced-Mid_EYFP
FEATURES             Location/Qualifiers
     source          1..4421
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      281..290
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(287..289,290..292,293..1006)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(332..353)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(593..612)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     940..961
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1051..1068)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1057..1281
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1327..1755
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1414..1433)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1624..1645
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1778..1974
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1825..1960
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1887..1906
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2094..2215
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2131..2150)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2185..2204
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2264..2280)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2264..2280)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2277..2299)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2288..2304
                     /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(2312..2318,2319..2336,2337..2342))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2357..2378
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2495..2512)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2666..3254)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2746..2765)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3425..4216,4217..4285))
                     /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     4048..4067
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4286..4390)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctgaa ttcggatcca gagtgaggac tacctataga agtaagagtg
       61 aggacaatcg tatacaagta gatccagagt gaggactacc tatagaagta agagtgagga
      121 caatcgtata caagtagatc cctctagagg gagatcccat gaagaatctg cttagggtta
      181 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gatccctcta gagggtatat
      241 aatgggggcc actagtctac taccagagct catcgctagc gccaccatgg tgagcaaggg
      301 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
      361 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
      421 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt
      481 cggctacggc ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt
      541 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
      601 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
      661 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
      721 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
      781 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
      841 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca
      901 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
      961 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaggcgg ccgctcgagt
     1021 ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     1081 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     1141 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     1201 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     1261 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     1321 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1381 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1441 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1501 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1561 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1621 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1681 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1741 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta ggctcaatta gtcagcaacc
     1801 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     1861 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     1921 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     1981 ccgggagctt gtatatccat tttcggaggt tgggcttcgg aatcgttttc cgggacgccg
     2041 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     2101 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     2161 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     2221 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     2281 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     2341 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     2401 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     2461 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2521 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2581 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2641 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2701 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2761 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2821 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2881 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2941 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3001 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3061 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     3121 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3181 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3241 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3301 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3361 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3421 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3481 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3541 gccccagtgc tgcaatgata ccgcgagatc cacgctcacc ggctccagat ttatcagcaa
     3601 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3661 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3721 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3781 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3841 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3901 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3961 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4021 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4081 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4141 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4201 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4261 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     4321 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4381 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
