LOCUS       sequence-365452-        6413 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 6413)
  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 6413)
  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.14
FEATURES             Location/Qualifiers
     source          1..6413
                     /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"
     promoter        299..576
                     /label=chicken beta-actin promoter
     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      849..858
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(855..857,858..860,861..1574)
                     /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(900..921)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1508..1529
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1605..2179,2180..2182,2183..2191)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1772..1789)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1999..2018
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     ncRNA           3115..3129
                     /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(3435..3454)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3500..3555
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3554..3573)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3916..3932)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3916..3932)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3929..3951)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3940..3956
                     /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(3964..3970,3971..3988,3989..3994))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4009..4030
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4089..4284
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4135..4270
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4290..4424
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4327..4346)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4381..4400
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4492..4509)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4663..5251)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4743..4762)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5422..6213,6214..6282))
                     /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     6045..6064
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6283..6387)
                     /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 gggcgaggcg grgaggtgcg
      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 gccgaggggt atactcccct
      841 cggcggccgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      901 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtccgcggc gagggcgagg
      961 gcgatgccac caacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     1021 tgccctggcc caccctcgtg accaccttcg gctacggcgt ggcctgcttc agccgctacc
     1081 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     1141 agcgcaccat ctctttcaag gacgacggta cctacaagac ccgcgccgag gtgaagttcg
     1201 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1261 acatcctggg gcacaagctg gagtacaact tcaacagcca ctacgtctat atcacggccg
     1321 acaagcagaa gaactgcatc aaggctaact tcaagatccg ccacaacgtt gaggacggca
     1381 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1441 tgcccgacaa ccactacctg agccatcagt ccaagctgag caaagacccc aacgagaagc
     1501 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gattacacat ggcatggacg
     1561 agctgtacaa gtagcctgca gggtcaccta aataattccg cccccccctc tccctccccc
     1621 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg
     1681 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc
     1741 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg
     1801 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg
     1861 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca
     1921 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata
     1981 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc
     2041 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt
     2101 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt
     2161 tgaaaaacac gatgataata tggccacaac catgggctgc accgtgagcg ccgaggacaa
     2221 ggcggcggcc gagcgcggct ctggttccgg cactagtgtg agtaaaggag aggccgttat
     2281 aaaagagttc atgcggttca aggtacacat ggagggatca atgaacggac acgagttcga
     2341 aatcgaaggg gaaggagagg gcaggcccta tgagggtact cagaccgcca aactcaaagt
     2401 gacaaaggga ggccctttgc ctttctcttg ggacatcttg tcaccacaat ttatgtatgg
     2461 ctcccgagca tttataaaac acccagcaga catacccgat tactacaagc agtctttccc
     2521 agaaggtttt aaatgggaaa gggtcatgaa ctttgaggat ggtggtgctg tgacggtgac
     2581 gcaggatact tctttggagg acggaacact catctacaag gtaaagctca ggggcacgaa
     2641 cttcccgccg gacggaccag tcatgcagaa aaagactatg ggatgggagg cttccaccga
     2701 acggttgtat cccgaggacg gtgttttgaa gggagatata aaaatggctc ttcggctgaa
     2761 agatggaggc agatacttgg cagattttaa gaccacgtat aaggcaaaaa aaccagtgca
     2821 aatgccggga gcatataacg ttgaccgcaa actggacatc acctcacata acgaggatta
     2881 tacagtagta gaacagtacg aaagaagcga agggaggcac agcaccgggg gtatggatga
     2941 gctgtataag tgataggcat gctgtttgaa tgaggcttca gtactttaca gaatcgttgc
     3001 ctgcacatct tggaaacact tgctgggatt acttcgactt cttaacccaa cagaaggctc
     3061 gagaaggtat attgctgttg acagtgagcg cactctttct aggaggttgt gatagtgaag
     3121 ccacagatgt atcacaacct cctagaaaga gtatgcctac tgcctcggac ttcaaggggc
     3181 tagaattcga gcaattatct tgtttactaa aactgaatac cttgctatct ctttgataca
     3241 tttttacaaa gctgaattaa aatggtataa attaaatcac ttttttcaat tgacgcgtga
     3301 tctaattcta ccgggtaggg gaggcgcttt tcccaaggca gtctggagca tggccgcact
     3361 cctcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc cctggctcac
     3421 aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt
     3481 gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat
     3541 tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat
     3601 gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat gaacaaaggt
     3661 tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat
     3721 agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct
     3781 ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga
     3841 ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct gcagcccaag
     3901 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     3961 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4021 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4081 gcggatccgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     4141 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     4201 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     4261 tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca
     4321 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4381 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat
     4441 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4501 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4561 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4621 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4681 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4741 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4801 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4861 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4921 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4981 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5041 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5101 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5161 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5221 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5281 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5341 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5401 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5461 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5521 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5581 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5641 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5701 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5761 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5821 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5881 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5941 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6001 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6061 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6121 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6181 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6241 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6301 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6361 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctg
//
