LOCUS       sequence-365376-        6820 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  One in a series of plasmids to titrate levels of expressed protein 
            to levels of endogenous protein (attenuated EF1a promoter, Fig 3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6820)
  AUTHORS   Millette MM, Holland ED, Tenpas TJ, Dent EW
  TITLE     A Single Transcript Knockdown-Replacement Strategy Employing 5' UTR 
            Secondary Structures to Precisely Titrate Rescue Protein 
            Translation.
  JOURNAL   Front Genome Ed. 2022 Mar 28;4:803375. doi: 
            10.3389/fgeed.2022.803375. eCollection 2022.
  PUBMED    35419562
REFERENCE   2  (bases 1 to 6820)
  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: "Front 
            Genome Ed."; date: "2022-03-28"; volume: "4:803375. doi"; pages: " 
            10.3389/fgeed.2022.803375. eCollection 2022"
COMMENT     Sequence Label: pKDR-EF1a.4
FEATURES             Location/Qualifiers
     source          1..6820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        36..1218
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          267..1209
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1166..1186
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1256..1265
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1262..1264,1265..1267,1268..1981)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1307..1328)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1915..1936
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(2012..2586,2587..2589,2590..2598)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2179..2196)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2406..2425
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     ncRNA           3522..3536
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     primer_bind     complement(3842..3861)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3907..3962
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3961..3980)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4323..4339)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4323..4339)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4336..4358)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4347..4363
                     /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(4371..4377,4378..4395,4396..4401))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4416..4437
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4496..4691
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4542..4677
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4697..4831
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4734..4753)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4788..4807
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4899..4916)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5070..5658)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5150..5169)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5829..6620,6621..6689))
                     /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     6452..6471
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6690..6794)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagatcatcg cgtgaggctc cggtgcccgt cagtgggcag
       61 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaagcggtg
      121 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
      181 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
      241 gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc
      301 tctttacggg ttatggccct tgcgtgcctt gaattacttc cacgcccctg gctgcagtac
      361 gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct
      421 taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc
      481 gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
      541 taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg
      601 ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt
      661 gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga
      721 cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc
      781 gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg
      841 ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg
      901 gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt
      961 gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctgcag cttttggagt
     1021 acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg
     1081 gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt
     1141 tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt
     1201 ccatttcagg tgtcgtgagg accggtcggc cgcagcccta tactgggctg cggccgccac
     1261 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1321 cggcgacgta aacggccaca agttcagcgt ccgcggcgag ggcgagggcg atgccaccaa
     1381 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1441 cctcgtgacc accttcggct acggcgtggc ctgcttcagc cgctaccccg accacatgaa
     1501 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctc
     1561 tttcaaggac gacggtacct acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1621 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1681 caagctggag tacaacttca acagccacta cgtctatatc acggccgaca agcagaagaa
     1741 ctgcatcaag gctaacttca agatccgcca caacgttgag gacggcagcg tgcagctcgc
     1801 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1861 ctacctgagc catcagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt
     1921 cctgctggag ttcgtgaccg ccgccgggat tacacatggc atggacgagc tgtacaagta
     1981 gcctgcaggg tcacctaaat aattccgccc ccccctctcc ctcccccccc cctaacgtta
     2041 ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca
     2101 tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca
     2161 ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg
     2221 aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc
     2281 agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata
     2341 cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag
     2401 tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc
     2461 attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt
     2521 taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat
     2581 gataatatgg ccacaaccat gggctgcacc gtgagcgccg aggacaaggc ggcggccgag
     2641 cgcggctctg gttccggcac tagtgtgagt aaaggagagg ccgttataaa agagttcatg
     2701 cggttcaagg tacacatgga gggatcaatg aacggacacg agttcgaaat cgaaggggaa
     2761 ggagagggca ggccctatga gggtactcag accgccaaac tcaaagtgac aaagggaggc
     2821 cctttgcctt tctcttggga catcttgtca ccacaattta tgtatggctc ccgagcattt
     2881 ataaaacacc cagcagacat acccgattac tacaagcagt ctttcccaga aggttttaaa
     2941 tgggaaaggg tcatgaactt tgaggatggt ggtgctgtga cggtgacgca ggatacttct
     3001 ttggaggacg gaacactcat ctacaaggta aagctcaggg gcacgaactt cccgccggac
     3061 ggaccagtca tgcagaaaaa gactatggga tgggaggctt ccaccgaacg gttgtatccc
     3121 gaggacggtg ttttgaaggg agatataaaa atggctcttc ggctgaaaga tggaggcaga
     3181 tacttggcag attttaagac cacgtataag gcaaaaaaac cagtgcaaat gccgggagca
     3241 tataacgttg accgcaaact ggacatcacc tcacataacg aggattatac agtagtagaa
     3301 cagtacgaaa gaagcgaagg gaggcacagc accgggggta tggatgagct gtataagtga
     3361 taggcatgct gtttgaatga ggcttcagta ctttacagaa tcgttgcctg cacatcttgg
     3421 aaacacttgc tgggattact tcgacttctt aacccaacag aaggctcgag aaggtatatt
     3481 gctgttgaca gtgagcgcac tctttctagg aggttgtgat agtgaagcca cagatgtatc
     3541 acaacctcct agaaagagta tgcctactgc ctcggacttc aaggggctag aattcgagca
     3601 attatcttgt ttactaaaac tgaatacctt gctatctctt tgatacattt ttacaaagct
     3661 gaattaaaat ggtataaatt aaatcacttt tttcaattga cgcgtgatct aattctaccg
     3721 ggtaggggag gcgcttttcc caaggcagtc tggagcatgg ccgcactcct caggtgcagg
     3781 ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag
     3841 atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt
     3901 ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct
     3961 cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt
     4021 agagtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag
     4081 gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga
     4141 cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa
     4201 aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca
     4261 tagctgtccc tcttctctta tggagatccc tcgacctgca gcccaagctt ggcgtaatca
     4321 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4381 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4441 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc
     4501 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     4561 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4621 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4681 gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4741 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4801 actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg
     4861 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4921 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4981 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5041 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5101 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5161 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5221 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5281 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5341 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5401 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5461 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5521 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5581 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5641 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5701 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5761 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5821 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5881 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5941 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6001 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6061 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6121 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6181 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6241 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6301 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6361 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6421 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6481 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6541 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6601 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6661 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6721 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6781 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
//
