LOCUS       sequence-365458-        6411 bp    DNA     circular SYN 15-APR-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 6411)
  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 6411)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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.6
FEATURES             Location/Qualifiers
     source          1..6411
                     /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      847..856
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(853..855,856..858,859..1572)
                     /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(898..919)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1506..1527
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1603..2177,2178..2180,2181..2189)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1770..1787)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1997..2016
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     ncRNA           3113..3127
                     /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(3433..3452)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3498..3553
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3552..3571)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3914..3930)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3914..3930)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3927..3949)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3938..3954
                     /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(3962..3968,3969..3986,3987..3992))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4007..4028
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4087..4282
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4133..4268
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4288..4422
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4325..4344)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4379..4398
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4490..4507)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4661..5249)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4741..4760)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5420..6211,6212..6280))
                     /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     6043..6062
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6281..6385)
                     /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 gggsgaggcg gagaggkgcg
      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 gcgtgcggta tactccgcac
      841 gcggccgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      901 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tccgcggcga gggcgagggc
      961 gatgccacca acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1021 ccctggccca ccctcgtgac caccttcggc tacggcgtgg cctgcttcag ccgctacccc
     1081 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1141 cgcaccatct ctttcaagga cgacggtacc tacaagaccc gcgccgaggt gaagttcgag
     1201 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1261 atcctggggc acaagctgga gtacaacttc aacagccact acgtctatat cacggccgac
     1321 aagcagaaga actgcatcaa ggctaacttc aagatccgcc acaacgttga ggacggcagc
     1381 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1441 cccgacaacc actacctgag ccatcagtcc aagctgagca aagaccccaa cgagaagcgc
     1501 gatcacatgg tcctgctgga gttcgtgacc gccgccggga ttacacatgg catggacgag
     1561 ctgtacaagt agcctgcagg gtcacctaaa taattccgcc cccccctctc cctccccccc
     1621 ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt
     1681 attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
     1741 cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
     1801 tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac
     1861 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg
     1921 tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt
     1981 tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca
     2041 gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt
     2101 ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg
     2161 aaaaacacga tgataatatg gccacaacca tgggctgcac cgtgagcgcc gaggacaagg
     2221 cggcggccga gcgcggctct ggttccggca ctagtgtgag taaaggagag gccgttataa
     2281 aagagttcat gcggttcaag gtacacatgg agggatcaat gaacggacac gagttcgaaa
     2341 tcgaagggga aggagagggc aggccctatg agggtactca gaccgccaaa ctcaaagtga
     2401 caaagggagg ccctttgcct ttctcttggg acatcttgtc accacaattt atgtatggct
     2461 cccgagcatt tataaaacac ccagcagaca tacccgatta ctacaagcag tctttcccag
     2521 aaggttttaa atgggaaagg gtcatgaact ttgaggatgg tggtgctgtg acggtgacgc
     2581 aggatacttc tttggaggac ggaacactca tctacaaggt aaagctcagg ggcacgaact
     2641 tcccgccgga cggaccagtc atgcagaaaa agactatggg atgggaggct tccaccgaac
     2701 ggttgtatcc cgaggacggt gttttgaagg gagatataaa aatggctctt cggctgaaag
     2761 atggaggcag atacttggca gattttaaga ccacgtataa ggcaaaaaaa ccagtgcaaa
     2821 tgccgggagc atataacgtt gaccgcaaac tggacatcac ctcacataac gaggattata
     2881 cagtagtaga acagtacgaa agaagcgaag ggaggcacag caccgggggt atggatgagc
     2941 tgtataagtg ataggcatgc tgtttgaatg aggcttcagt actttacaga atcgttgcct
     3001 gcacatcttg gaaacacttg ctgggattac ttcgacttct taacccaaca gaaggctcga
     3061 gaaggtatat tgctgttgac agtgagcgca ctctttctag gaggttgtga tagtgaagcc
     3121 acagatgtat cacaacctcc tagaaagagt atgcctactg cctcggactt caaggggcta
     3181 gaattcgagc aattatcttg tttactaaaa ctgaatacct tgctatctct ttgatacatt
     3241 tttacaaagc tgaattaaaa tggtataaat taaatcactt ttttcaattg acgcgtgatc
     3301 taattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg gccgcactcc
     3361 tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa
     3421 ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga
     3481 gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt
     3541 tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga
     3601 gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg
     3661 gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag
     3721 aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt
     3781 aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact
     3841 actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc agcccaagct
     3901 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     3961 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4021 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4081 ggatccgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     4141 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     4201 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     4261 tggaggccta ggcttttgca aaaagctaac ttgtttattg cagcttataa tggttacaaa
     4321 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4381 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tccgctgcat taatgaatcg
     4441 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4501 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4561 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4621 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4681 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4741 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4801 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4861 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4921 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4981 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5041 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5101 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5161 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5221 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5281 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5341 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5401 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5461 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5521 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5581 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5641 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5701 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5761 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5821 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5881 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5941 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6001 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6061 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6121 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6181 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6241 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6301 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6361 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct g
//
