LOCUS       sequence-369891-        6958 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of Cluc-Gluc reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6958)
  AUTHORS   Kato K, Zhou W, Okazaki S, Isayama Y, Nishizawa T, Gootenberg JS, 
            Abudayyeh OO, Nishimasu H
  TITLE     Structure and engineering of the type III-E CRISPR-Cas7-11 effector 
            complex.
  JOURNAL   Cell. 2022 May 25. pii: S0092-8674(22)00581-5. doi: 
            10.1016/j.cell.2022.05.003.
  PUBMED    35643083
REFERENCE   2  (bases 1 to 6958)
  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: "Cell. 2022 
            May 25. pii: S0092-8674(22)00581-5. doi: 
            10.1016/j.cell.2022.05.003."
COMMENT     Sequence Label: pDF0112
            cmv-cluciferase-polya-ef1a-g-luciferas...
FEATURES             Location/Qualifiers
     source          1..6958
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     19..36
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    153..174
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(189..194,195..212,213..219)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    227..243
                     /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)."
     primer_bind     232..254
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     251..267
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     251..267
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    281..329
                     /note="synthetic polyadenylation signal"
     CDS             complement(330..1991)
                     /codon_start=1
                     /product="secreted Cypridina luciferase"
                     /label=CLuc
                     /note="contains 30 codon substitutions for efficient 
                     translation in mammalian cells"
                     /translation="MKTLILAVALVYCATVHCQDCPYEPDPPNTVPTSCEAKEGECIDS
                     SCGTCTRDILSDGLCENKPGKTCCRMCQYVIECRVEAAGWFRTFYGKRFQFQEPGTYVL
                     GQGTKGGDWKVSITLENLDGTKGAVLTKTRLEVAGDIIDIAQATENPITVNGGADPIIA
                     NPYTIGEVTIAVVEMPGFNITVIEFFKLIVIDILGGRSVRIAPDTANKGMISGLCGDLK
                     MMEDTDFTSDPEQLAIQPKINQEFDGCPLYGNPDDVAYCKGLLEPYKDSCRNPINFYYY
                     TISCAFARCMGGDERASHVLLDYRETCAAPETRGTCVLSGHTFYDTFDKARYQFQGPCK
                     EILMAADCFWNTWDVKVSHRNVDSYTEVEKVRIRKQSTVVELIVDGKQILVGGEAVSVP
                     YSSQNTSIYWQDGDILTTAILPEALVVKFNFKQLLVVHIRDPFDGKTCGICGNYNQDFS
                     DDSFDAEGACDLTPNPPGCTEEQKPEAERLCNSLFAGQSDLDQKCNVCHKPDRVERCMY
                     EYCLRGQQGFCDHAWEFKKECYIKHGDTLEVPDECK"
     primer_bind     complement(2016..2035)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2017..2035)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2080..2283)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2109..2129)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2284..2663
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2777..3955
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3008..3946
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3903..3923
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3974..3983
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3980..4537
                     /codon_start=1
                     /product="secreted Gaussia luciferase"
                     /label=hGLuc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGGD"
     polyA_signal    4538..4586
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4609..4626)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4609..4625)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4618..4640)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4834..4853)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4953..4975
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5013..5031)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5099..5203
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5204..5272,5273..6064)
                     /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     complement(5422..5441)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6235..6823
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
       61 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
      121 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
      181 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
      241 caatttcaca caggaaacag ctatgaccat gattacgcca cacacaaaaa accaacacac
      301 agatgtaatg aaaataaaga tattttattt tatttgcatt catctggtac ttctagggtg
      361 tctccatgct ttatgtagca ttctttcttg aactcccatg cgtggtcaca gaaaccctgt
      421 tgtcccctca ggcaatactc gtacatgcat cgttcgacac ggtcaggctt gtggcacacg
      481 ttacatttct gatcaagatc actttgaccg gcgaagagac tattgcagag tcgttcagct
      541 tcaggtttct gttcttcggt gcatcccggt gggttggggg tcagatcaca ggctccttca
      601 gcatcaaaag aatcatcact gaaatcctgg ttgtagttac cgcaaatacc gcaagtctta
      661 ccatcgaatg gatctctaat atgtacgacg agcagttgct tgaagttgaa cttgaccacc
      721 agagcttcag gtaggatggc tgtagtcagt atgtcaccat cttgccagta gatggaagtg
      781 ttctgagagc tgtacgggac ggacacggct tctcctccaa ccagaatctg ttttccatca
      841 acaatgagtt ctactacagt cgattgtttc ctgattcgta ctttctctac ttcagtgtaa
      901 gagtcaacat tcctgtgtga aaccttcaca tcccaagtgt tccagaaaca gtcggcggcc
      961 ataagaatct ccttgcaggg accctggaat tggtatcttg ctttgtcaaa tgtatcgtag
     1021 aaagtatgtc cagacaaaac gcaggttcct ctagtttcgg gagcagcgca cgtctccctg
     1081 tagtcaagca gcacgtgtga ggctcgctcg tctccaccca tacagcgggc gaaggcgcag
     1141 gagatggtgt agtagtagaa gttgatgggg ttgcggcagc tgtccttgta cggctccaga
     1201 agacctttgc agtatgcaac gtcatcagga tttccataga gtggacaacc gtcaaactcc
     1261 tggttgatct taggctgaat agcgagttgt tctggatctg aagtgaagtc tgtatcttcc
     1321 atcattttaa gatctccaca gaggccagag atcattcctt tgtttgctgt gtctggggcg
     1381 attcttacag atcttcctcc gaggatgtcg atcacgatca gtttgaagaa ctcaatgacg
     1441 gtgatgttga agcctggcat ctcaacaaca gcgatggtga cctcgccgat ggtgtacggg
     1501 ttggcgatga tagggtcagc tccaccgttt acagtgatgg gattctcagt agcttgagcg
     1561 atgtcaatga tgtctccagc cacttccagt cttgtcttgg tcagcacagc ccccttggtt
     1621 ccatccaggt tctccagggt gatggacacc ttccagtcgc cgcccttggt tccttgaccc
     1681 aacacgtatg taccaggttc ctggaactgg aatctctttc catagaatgt tctaaaccat
     1741 cctgcggcct ctactctgca ttcaattaca tactgacaca ttcgacaaca tgtttttcct
     1801 ggtttatttt cacacagtcc atctgatagt atgtctctcg tgcaggtgcc acagctgcta
     1861 tcaatacatt ctccttcttt agcttcacag gaagttggaa ctgtgtttgg tggatcaggt
     1921 tcgtaaggac agtcctggca atgaacagtg gcgcagtaga ctaatgcaac ggcaagaatt
     1981 aaggtcttca tggtggcggt accaagcttg ggtctcccta tagtgagtcg tattaatttc
     2041 gataagccag taagcagtgg gttctctagt tagccagaga gctctgctta tatagacctc
     2101 ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg gagttgttac gacattttgg
     2161 aaagtcccgt tgattttggt gccaaaacaa actcccattg acgtcaatgg ggtggagact
     2221 tggaaatccc cgtgagtcaa accgctatcc acgcccattg atgtactgcc aaaaccgcat
     2281 caccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa agtcccataa
     2341 ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg
     2401 cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc gtaaatactc
     2461 cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat acgtcattat
     2521 tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta ccgtaagtta
     2581 tgtaacgcgg aactccatat atgggctatg aactaatgac cccgtaattg attactatta
     2641 ataactagtc aataatcaat gtcaacgcgt atatctggcc cgtacatcga agctgatgca
     2701 aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct tctaggtctt
     2761 gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt
     2821 ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg
     2881 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga
     2941 accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag
     3001 aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc
     3061 ttgcgtgcct tgaattactt ccacctggct gcagtacgtg attcttgatc ccgagcttcg
     3121 ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc
     3181 ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg
     3241 cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc
     3301 gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat
     3361 ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc
     3421 gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg
     3481 gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct
     3541 ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg
     3601 gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag
     3661 gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc
     3721 gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga
     3781 ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc
     3841 ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat
     3901 tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgacgtac
     3961 gggatcctct agagccacca tgggagtcaa agttctgttt gccctgatct gcatcgctgt
     4021 ggccgaggcc aagcccaccg agaacaacga agacttcaac atcgtggccg tggccagcaa
     4081 cttcgcgacc acggatctcg atgctgaccg cgggaagttg cccggcaaga agctgccgct
     4141 ggaggtgctc aaagagatgg aagccaatgc ccggaaagct ggctgcacca ggggctgtct
     4201 gatctgcctg tcccacatca agtgcacgcc caagatgaag aagttcatcc caggacgctg
     4261 ccacacctac gaaggcgaca aagagtccgc acagggcggc ataggcgagg cgatcgtcga
     4321 cattcctgag attcctgggt tcaaggactt ggagcccatg gagcagttca tcgcacaggt
     4381 cgatctgtgt gtggactgca caactggctg cctcaaaggg cttgccaacg tgcagtgttc
     4441 tgacctgctc aagaagtggc tgccgcaacg ctgtgcgacc tttgccagca agatccaggg
     4501 ccaggtggac aagatcaagg gggccggtgg tgactaaaat aaaatatctt tattttcatt
     4561 acatctgtgt gttggttttt tgtgtgcccg ggtaccgagc tcgaattcac tggccgtcgt
     4621 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     4681 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     4741 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     4801 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4861 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4921 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4981 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     5041 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5101 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5161 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5221 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5281 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5341 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5401 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5461 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5521 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5581 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5641 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5701 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5761 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     5821 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5881 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     5941 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6001 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6061 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6121 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6181 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6241 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6301 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6361 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6421 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6481 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6541 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6601 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6661 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6721 agggggaaac gcctggtatm tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6781 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     6841 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     6901 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgc
//
