LOCUS       sequence-323339-        4337 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EYFP reporter vector for the ZF43x4-Spaced promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4337)
  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 4337)
  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: pPD064 ZF43x4-Spaced_EYFP
FEATURES             Location/Qualifiers
     source          1..4337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      228..237
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(234..236,237..239,240..953)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(279..300)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(540..559)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     887..908
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(998..1015)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1004..1228
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1274..1702
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1361..1380)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1571..1592
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1725..1921
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1772..1907
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1834..1853
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2041..2162
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2078..2097)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2132..2151
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2211..2227)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2211..2227)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2224..2246)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2235..2251
                     /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(2259..2265,2266..2283,2284..2289))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2304..2325
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2442..2459)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2613..3201)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2693..2712)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3372..4163,4164..4232))
                     /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     3995..4014
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4233..4337)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctga attcggatcc
       61 agagtgagga ctacctatag aagtaagagt gaggacaatc gtatacaagt agatccagag
      121 tgaggactac ctatagaagt aagagtgagg acaatcgtat acaagtagat ccctctagag
      181 ggtatataat gggggccact agtctactac cagagctcat cgctagcgcc accatggtga
      241 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
      301 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
      361 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
      421 ccaccttcgg ctacggcctg cagtgcttcg cccgctaccc cgaccacatg aagcagcacg
      481 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
      541 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
      601 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
      661 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
      721 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
      781 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
      841 gctaccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
      901 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taggcggccg
      961 ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     1021 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     1081 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     1141 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     1201 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     1261 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1321 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1381 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1441 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1501 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1561 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     1621 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     1681 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggc tcaattagtc
     1741 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     1801 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     1861 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     1921 aaagctcccg ggagcttgta tatccatttt cggaggttgg gcttcggaat cgttttccgg
     1981 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     2041 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     2101 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     2161 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     2221 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     2281 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     2341 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     2401 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     2461 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     2521 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     2581 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     2641 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2701 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2761 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     2821 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2881 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     2941 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3001 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     3061 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3121 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3181 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3241 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     3301 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     3361 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     3421 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     3481 ccatctggcc ccagtgctgc aatgataccg cgagatccac gctcaccggc tccagattta
     3541 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     3601 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     3661 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     3721 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     3781 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     3841 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     3901 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     3961 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4021 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4081 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4141 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4201 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     4261 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     4321 caaatagggg ttccgcg
//
