LOCUS       sequence-323337-        4281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EYFP reporter vector for the ZF43x2-Spaced promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4281)
  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 4281)
  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: pPD062 ZF43x2-Spaced_EYFP
FEATURES             Location/Qualifiers
     source          1..4281
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      172..181
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(178..180,181..183,184..897)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(223..244)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(484..503)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     831..852
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(942..959)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    948..1172
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1218..1646
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1305..1324)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1515..1536
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1669..1865
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1716..1851
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1778..1797
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    1985..2106
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2022..2041)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2076..2095
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2155..2171)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2155..2171)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2168..2190)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2179..2195
                     /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(2203..2209,2210..2227,2228..2233))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2248..2269
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2386..2403)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2557..3145)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2637..2656)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3316..4107,4108..4176))
                     /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     3939..3958
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4177..4281)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctga attcggatcc
       61 agagtgagga ctacctatag aagtaagagt gaggacaatc gtatacaagt agatccctct
      121 agagggtata taatgggggc cactagtcta ctaccagagc tcatcgctag cgccaccatg
      181 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      241 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
      301 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
      361 gtgaccacct tcggctacgg cctgcagtgc ttcgcccgct accccgacca catgaagcag
      421 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
      481 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
      541 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
      601 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
      661 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
      721 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
      781 ctgagctacc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
      841 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaggcg
      901 gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
      961 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     1021 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     1081 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     1141 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     1201 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     1261 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     1321 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     1381 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     1441 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     1501 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     1561 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     1621 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt aggctcaatt
     1681 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     1741 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     1801 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     1861 gcaaaaagct cccgggagct tgtatatcca ttttcggagg ttgggcttcg gaatcgtttt
     1921 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     1981 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     2041 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     2101 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     2161 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     2221 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     2281 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     2341 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     2401 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2461 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2521 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2581 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2641 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2701 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     2761 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2821 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2881 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2941 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3001 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3061 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3121 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3181 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3241 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3301 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3361 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3421 cttaccatct ggccccagtg ctgcaatgat accgcgagat ccacgctcac cggctccaga
     3481 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3541 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3601 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3661 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3721 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3781 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     3841 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     3901 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3961 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4021 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4081 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4141 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4201 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4261 taaacaaata ggggttccgc g
//
