LOCUS       sequence-323527-        4477 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EYFP reporter vector for the ZF43x6-Spaced-Mid promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4477)
  TITLE     Leonard Lab COMET plasmids
REFERENCE   2  (bases 1 to 4477)
  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"
COMMENT     Sequence Label: pPD136 ZF43x6-Spaced-Mid_EYFP
FEATURES             Location/Qualifiers
     source          1..4477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      337..346
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(343..345,346..348,349..1062)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(388..409)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(649..668)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     996..1017
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1107..1124)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1113..1337
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1383..1811
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1470..1489)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1680..1701
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1834..2030
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1881..2016
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1943..1962
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2150..2271
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2187..2206)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2241..2260
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2320..2336)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2320..2336)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2333..2355)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2344..2360
                     /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(2368..2374,2375..2392,2393..2398))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2413..2434
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2551..2568)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2722..3310)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2802..2821)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3481..4272,4273..4341))
                     /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     4104..4123
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4342..4446)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctgaa ttcggatcca gagtgaggac tacctataga agtaagagtg
       61 aggacaatcg tatacaagta gatccagagt gaggactacc tatagaagta agagtgagga
      121 caatcgtata caagtagatc cagagtgagg actacctata gaagtaagag tgaggacaat
      181 cgtatacaag tagatccctc tagagggaga tcccatgaag aatctgctta gggttaggcg
      241 ttttgcgctg cttcgcgatg tacgggccag atatacgatc cctctagagg gtatataatg
      301 ggggccacta gtctactacc agagctcatc gctagcgcca ccatggtgag caagggcgag
      361 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
      421 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
      481 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcggc
      541 tacggcctgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
      601 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
      661 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
      721 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
      781 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
      841 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
      901 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc
      961 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1021 gccgccggga tcactctcgg catggacgag ctgtacaagt aggcggccgc tcgagtctag
     1081 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     1141 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     1201 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     1261 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     1321 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     1381 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1441 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1501 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1561 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     1621 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     1681 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     1741 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     1801 taacgcgaat taattctgtg gaatgtgtgt cagttaggct caattagtca gcaaccatag
     1861 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     1921 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     1981 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     2041 gagcttgtat atccattttc ggaggttggg cttcggaatc gttttccggg acgccggctg
     2101 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     2161 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2221 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     2281 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     2341 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     2401 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     2461 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     2521 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     2581 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     2641 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     2701 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     2761 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     2821 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     2881 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     2941 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     3001 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     3061 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     3121 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     3181 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     3241 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     3301 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     3361 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     3421 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     3481 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     3541 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     3601 cagtgctgca atgataccgc gagatccacg ctcaccggct ccagatttat cagcaataaa
     3661 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     3721 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     3781 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     3841 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     3901 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     3961 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     4021 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     4081 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     4141 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     4201 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     4261 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     4321 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     4381 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     4441 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
