LOCUS       sequence-365454-        6409 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 CBh promoter, Fig 3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6409)
  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 6409)
  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-CBh.4
FEATURES             Location/Qualifiers
     source          1..6409
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        12..297
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          577..804
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      845..854
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(851..853,854..856,857..1570)
                     /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(896..917)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1504..1525
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1601..2175,2176..2178,2179..2187)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1768..1785)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1995..2014
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     ncRNA           3111..3125
                     /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(3431..3450)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3496..3551
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3550..3569)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3912..3928)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3912..3928)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3925..3947)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3936..3952
                     /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(3960..3966,3967..3984,3985..3990))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4005..4026
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4085..4280
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4131..4266
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4286..4420
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4323..4342)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4377..4396
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4488..4505)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4659..5247)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4739..4758)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5418..6209,6210..6278))
                     /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     6041..6060
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6279..6383)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacgtac ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
       61 ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg
      121 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      181 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc
      241 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
      301 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      361 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      421 cgcgcgccrg gsggggsggg gsggggsgrg gggsggggsg gggsgrggcg grgaggkgcg
      481 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      541 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
      601 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      661 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag
      721 aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac
      781 ctgcctgaaa tcactttttt tcaggttgga ccggtcggcc gcagccctat actgggctgc
      841 ggccgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
      901 cgagctggac ggcgacgtaa acggccacaa gttcagcgtc cgcggcgagg gcgagggcga
      961 tgccaccaac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1021 ctggcccacc ctcgtgacca ccttcggcta cggcgtggcc tgcttcagcc gctaccccga
     1081 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1141 caccatctct ttcaaggacg acggtaccta caagacccgc gccgaggtga agttcgaggg
     1201 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1261 cctggggcac aagctggagt acaacttcaa cagccactac gtctatatca cggccgacaa
     1321 gcagaagaac tgcatcaagg ctaacttcaa gatccgccac aacgttgagg acggcagcgt
     1381 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     1441 cgacaaccac tacctgagcc atcagtccaa gctgagcaaa gaccccaacg agaagcgcga
     1501 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatt acacatggca tggacgagct
     1561 gtacaagtag cctgcagggt cacctaaata attccgcccc cccctctccc tccccccccc
     1621 ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat
     1681 tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct
     1741 tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg
     1801 tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc
     1861 tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg
     1921 tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg
     1981 tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga
     2041 aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt
     2101 agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa
     2161 aaacacgatg ataatatggc cacaaccatg ggctgcaccg tgagcgccga ggacaaggcg
     2221 gcggccgagc gcggctctgg ttccggcact agtgtgagta aaggagaggc cgttataaaa
     2281 gagttcatgc ggttcaaggt acacatggag ggatcaatga acggacacga gttcgaaatc
     2341 gaaggggaag gagagggcag gccctatgag ggtactcaga ccgccaaact caaagtgaca
     2401 aagggaggcc ctttgccttt ctcttgggac atcttgtcac cacaatttat gtatggctcc
     2461 cgagcattta taaaacaccc agcagacata cccgattact acaagcagtc tttcccagaa
     2521 ggttttaaat gggaaagggt catgaacttt gaggatggtg gtgctgtgac ggtgacgcag
     2581 gatacttctt tggaggacgg aacactcatc tacaaggtaa agctcagggg cacgaacttc
     2641 ccgccggacg gaccagtcat gcagaaaaag actatgggat gggaggcttc caccgaacgg
     2701 ttgtatcccg aggacggtgt tttgaaggga gatataaaaa tggctcttcg gctgaaagat
     2761 ggaggcagat acttggcaga ttttaagacc acgtataagg caaaaaaacc agtgcaaatg
     2821 ccgggagcat ataacgttga ccgcaaactg gacatcacct cacataacga ggattataca
     2881 gtagtagaac agtacgaaag aagcgaaggg aggcacagca ccgggggtat ggatgagctg
     2941 tataagtgat aggcatgctg tttgaatgag gcttcagtac tttacagaat cgttgcctgc
     3001 acatcttgga aacacttgct gggattactt cgacttctta acccaacaga aggctcgaga
     3061 aggtatattg ctgttgacag tgagcgcact ctttctagga ggttgtgata gtgaagccac
     3121 agatgtatca caacctccta gaaagagtat gcctactgcc tcggacttca aggggctaga
     3181 attcgagcaa ttatcttgtt tactaaaact gaataccttg ctatctcttt gatacatttt
     3241 tacaaagctg aattaaaatg gtataaatta aatcactttt ttcaattgac gcgtgatcta
     3301 attctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatggc cgcactcctc
     3361 aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg gctcacaaat
     3421 accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc
     3481 atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt
     3541 tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt
     3601 atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc
     3661 tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa
     3721 aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa
     3781 catccctaaa attttcctta catgttttac tagccagatt tttcctcctc tcctgactac
     3841 tcccagtcat agctgtccct cttctcttat ggagatccct cgacctgcag cccaagcttg
     3901 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     3961 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4021 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg
     4081 atccgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4141 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4201 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4261 gaggcctagg cttttgcaaa aagctaactt gtttattgca gcttataatg gttacaaata
     4321 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4381 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc cgctgcatta atgaatcggc
     4441 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4501 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4561 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4621 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4681 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4741 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4801 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4861 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4921 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4981 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5041 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5101 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5161 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5221 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5281 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5341 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5401 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5461 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5521 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5581 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5641 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5701 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5761 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5821 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5881 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5941 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6001 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6061 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6121 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6181 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6241 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6301 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6361 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctg
//
