LOCUS       sequence-323528-        4549 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EYFP reporter vector for the ZF43x8-Spaced-Mid promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4549)
  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 4549)
  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: pPD137 ZF43x8-Spaced-Mid_EYFP
FEATURES             Location/Qualifiers
     source          1..4549
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      409..418
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(415..417,418..420,421..1134)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(460..481)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(721..740)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1068..1089
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1179..1196)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1185..1409
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1455..1883
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1542..1561)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1752..1773
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1906..2102
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1953..2088
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2015..2034
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2222..2343
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2259..2278)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2313..2332
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2392..2408)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2392..2408)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2405..2427)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2416..2432
                     /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(2440..2446,2447..2464,2465..2470))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2485..2506
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2623..2640)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2794..3382)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2874..2893)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3553..4344,4345..4413))
                     /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     4176..4195
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4414..4518)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctgaa ttcggatcca gagtgaggac tacctataga agtaagagtg
       61 aggacaatcg tatacaagta gatccagagt gaggactacc tatagaagta agagtgagga
      121 caatcgtata caagtagatc cctctagagg gagatccaga gtgaggacta cctatagaag
      181 taagagtgag gacaatcgta tacaagtaga tccagagtga ggactaccta tagaagtaag
      241 agtgaggaca atcgtataca agtagatccc tctagaggga gatcccatga agaatctgct
      301 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacga tccctctaga
      361 gggtatataa tgggggccac tagtctacta ccagagctca tcgctagcgc caccatggtg
      421 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      481 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
      541 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      601 accaccttcg gctacggcct gcagtgcttc gcccgctacc ccgaccacat gaagcagcac
      661 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
      721 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
      781 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
      841 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
      901 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
      961 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1021 agctaccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1081 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaggcggcc
     1141 gctcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     1201 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     1261 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     1321 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     1381 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     1441 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     1501 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     1561 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     1621 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     1681 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     1741 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     1801 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     1861 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg ctcaattagt
     1921 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     1981 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     2041 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     2101 aaaagctccc gggagcttgt atatccattt tcggaggttg ggcttcggaa tcgttttccg
     2161 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     2221 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     2281 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2341 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     2401 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     2461 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     2521 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     2581 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     2641 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2701 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2761 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2821 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2881 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2941 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3001 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3061 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3121 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3181 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     3241 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3301 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3361 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3421 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3481 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3541 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3601 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3661 accatctggc cccagtgctg caatgatacc gcgagatcca cgctcaccgg ctccagattt
     3721 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3781 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3841 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3901 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3961 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     4021 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4081 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     4141 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     4201 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     4261 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4321 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4381 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4441 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4501 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
