LOCUS       sequence-323461-        4361 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EYFP reporter vector for the ZF43x5-Spaced promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4361)
  TITLE     Leonard Lab COMET plasmids
REFERENCE   2  (bases 1 to 4361)
  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: pPD068 ZF43x5-Spaced_EYFP
FEATURES             Location/Qualifiers
     source          1..4361
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      252..261
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(258..260,261..263,264..977)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(303..324)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(564..583)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     911..932
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1022..1039)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1028..1252
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1298..1726
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1385..1404)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1595..1616
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1749..1945
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1796..1931
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1858..1877
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2065..2186
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2102..2121)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2156..2175
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2235..2251)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2235..2251)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2248..2270)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2259..2275
                     /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(2283..2289,2290..2307,2308..2313))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2328..2349
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2466..2483)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2637..3225)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2717..2736)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3396..4187,4188..4256))
                     /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     4019..4038
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4257..4361)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctga attagagtga
       61 ggacaatcgt atacaagtag atccagagtg aggactacct atagaagtaa gagtgaggac
      121 aatcgtatac aagtagatcc agagtgagga ctacctatag aagtaagagt gaggacaatc
      181 gtatacaagt agatccctct agagggtata taatgggggc cactagtcta ctaccagagc
      241 tcatcgctag cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      301 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
      361 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
      421 ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cctgcagtgc ttcgcccgct
      481 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
      541 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
      601 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
      661 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
      721 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
      781 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
      841 tgctgcccga caaccactac ctgagctacc agtccgccct gagcaaagac cccaacgaga
      901 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
      961 acgagctgta caagtaggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca
     1021 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     1081 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     1141 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     1201 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     1261 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     1321 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     1381 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     1441 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     1501 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     1561 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     1621 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     1681 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     1741 tgtgtcagtt aggctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     1801 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     1861 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     1921 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggagg
     1981 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     2041 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     2101 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     2161 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     2221 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     2281 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     2341 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     2401 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     2461 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2521 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2581 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2641 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2701 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2761 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2821 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2881 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2941 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3001 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3061 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3121 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3181 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3241 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3301 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     3361 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     3421 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3481 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagat
     3541 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3601 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3661 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3721 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3781 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3841 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3901 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3961 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4021 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4081 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     4141 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     4201 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     4261 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4321 tttgaatgta tttagaaaaa taaacaaata ggggttccgc g
//
