LOCUS       sequence-125547-        7384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR-Display Cypridina Luciferase-2A-Venus YFP ""normalizer,"" 
            transient format..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7384)
  AUTHORS   Shechner DM, Hacisuleyman E, Younger ST, Rinn JL
  TITLE     Multiplexable, locus-specific targeting of long RNAs with 
            CRISPR-Display.
  JOURNAL   Nat Methods. 2015 Jul;12(7):664-70. doi: 10.1038/nmeth.3433. Epub 
            2015 Jun 1.
  PUBMED    26030444
REFERENCE   2  (bases 1 to 7384)
  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"; doi: 
            "10.1038/nmeth.3433"; journalName: "Nat Methods"; date: "2015-07"; 
            volume: "12"; issue: "7"; pages: "664-70"
COMMENT     Sequence Label: pTrans_9xD3B_Cluc_Norm
FEATURES             Location/Qualifiers
     source          1..7384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1..792,793..861))
                     /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     624..643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(862..966)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1034..1052
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1090..1112)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1212..1231
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1425..1447
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1439..1456
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1440..1456
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1819..1857
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1854..1878
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1904..3562
                     /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"
     CDS             3587..3640
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(3647..3649,3650..3652,3653..4363)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3692..3713)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3953..3972)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4300..4321
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4373..4429
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4436..5035
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4436..4455)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4932..4952
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5062..5650
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5115..5135)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5533..5544)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5756..5989
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6061..6182
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6098..6117)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6152..6171
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6231..6247)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6231..6247)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6244..6266)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6255..6271
                     /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(6279..6285,6286..6303,6304..6309))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6324..6345
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6455..6472)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6626..7214)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6706..6725)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     1021 attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga
     1081 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     1141 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg
     1201 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
     1261 accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg
     1321 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     1381 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     1441 taaaacgacg gccagtgaat tcgagctcgg taccttctcg agctttgcgg taatctagat
     1501 cgcccgtccc ctaggcaaag ctctaatcta gatcgcccgt cccctaggaa aggaagcagc
     1561 cacatctaga tcgcccgtcc cctaggataa cgttagatct agatcgcccg tcccctaggg
     1621 tgaagtagtc tttgcggtaa tctagatcgc ccgtccccta ggcaaagctc taatctagat
     1681 cgcccgtccc ctaggaaagg aagcagccac atctagatcg cccgtcccct aggataacgt
     1741 tagatctaga tcgcccgtcc cctagggtga agtagtatct agatcgcccg tcccctaggc
     1801 cggatccacg tatgtcgagg taggcgtgta cggtgggagg cctatataag cagagctcgt
     1861 ttagtgaacc gtcagatcgc ctggaggtac cgccaccgcc accatgaaga ccttaattct
     1921 tgccgttgca ttagtctact gcgccactgt tcattgccag gactgtcctt acgaacctga
     1981 tccaccaaac acagttccaa cttcctgtga agctaaagaa ggagaatgta ttgatagcag
     2041 ctgtggcacc tgcacgagag acatactatc agatggactg tgtgaaaata aaccaggaaa
     2101 aacatgttgc cgaatgtgtc agtatgtaat tgaatgcaga gtagaggccg caggatggtt
     2161 tagaacattc tatggaaaga gattccagtt ccaggaacct ggtacatacg tgttgggtca
     2221 aggaaccaag ggcggcgact ggaaggtgtc catcaccctg gagaacctgg atggaaccaa
     2281 gggggctgtg ctgaccaaga caagactgga agtggctgga gacatcattg acatcgctca
     2341 agctactgag aatcccatca ctgtaaacgg tggagctgac cctatcatcg ccaacccgta
     2401 caccatcggc gaggtcacca tcgctgttgt tgagatgcca ggcttcaaca tcaccgtcat
     2461 tgagttcttc aaactgatcg tgatcgacat cctcggagga agatctgtaa gaatcgcccc
     2521 agacacagca aacaaaggaa tgatctctgg cctctgtgga gatcttaaaa tgatggaaga
     2581 tacagacttc acttcagatc cagaacaact cgctattcag cctaagatca accaggagtt
     2641 tgacggttgt ccactctatg gaaatcctga tgacgttgca tactgcaaag gtcttctgga
     2701 gccgtacaag gacagctgcc gcaaccccat caacttctac tactacacca tctcctgcgc
     2761 cttcgcccgc tgtatgggtg gagacgagcg agcctcacac gtgctgcttg actacaggga
     2821 gacgtgcgct gctcccgaaa ctagaggaac ctgcgttttg tctggacata ctttctacga
     2881 tacatttgac aaagcaagat accaattcca gggtccctgc aaggagattc ttatggccgc
     2941 cgactgtttc tggaacactt gggatgtgaa ggtttcacac aggaatgttg actcttacac
     3001 tgaagtagag aaagtacgaa tcaggaaaca atcgactgta gtagaactca ttgttgatgg
     3061 aaaacagatt ctggttggag gagaagccgt gtccgtcccg tacagctctc agaacacttc
     3121 catctactgg caagatggtg acatactgac tacagccatc ctacctgaag ctctggtggt
     3181 caagttcaac ttcaagcaac tgctcgtcgt acatattaga gatccattcg atggtaagac
     3241 ttgcggtatt tgcggtaact acaaccagga tttcagtgat gattcttttg atgctgaagg
     3301 agcctgtgat ctgaccccca acccaccggg atgcaccgaa gaacagaaac ctgaagctga
     3361 acgactctgc aatagtctct tcgccggtca aagtgatctt gatcagaaat gtaacgtgtg
     3421 ccacaagcct gaccgtgtcg aacgatgcat gtacgagtat tgcctgaggg gacaacaggg
     3481 tttctgtgac cacgcatggg agttcaagaa agaatgctac ataaagcatg gagacaccct
     3541 agaagtacca gatgaatgca aaggcagcac cagcggcggc agtggagagg gcagaggaag
     3601 tctgctaaca tgcggtgacg tcgaggagaa tcctggccca ggcagtatgg tgagcaaggg
     3661 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     3721 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     3781 gaagctgatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     3841 gggctacggc ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt
     3901 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     3961 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     4021 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     4081 ctacaacagc cacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa
     4141 cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag ctcgccgacc actaccagca
     4201 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca
     4261 gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     4321 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggcagcg gtgctacgaa
     4381 cttcagtctc ctgaaacagg ctggggatgt ggaggagaac cccggcccag gcagcatgac
     4441 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     4501 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     4561 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     4621 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     4681 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     4741 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     4801 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     4861 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     4921 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     4981 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctagtaacg
     5041 taccggttag taatgatcga caatcaacct ctggattaca aaatttgtga aagattgact
     5101 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     5161 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     5221 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     5281 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     5341 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     5401 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg
     5461 acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     5521 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     5581 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     5641 gcctccccgc ctggcgatgg taccgctaag cacaattcga gctcggtacc tttaagacca
     5701 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     5761 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg
     5821 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     5881 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     5941 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca
     6001 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg
     6061 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     6121 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     6181 tatcatgtct gggtttaaac ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg
     6241 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     6301 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     6361 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6421 gcggggagag gcggtttgcg tattgggccg cttcctcgct cactgactcg ctgcgctcgg
     6481 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     6541 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     6601 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     6661 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     6721 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     6781 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     6841 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6901 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6961 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     7021 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     7081 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     7141 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     7201 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     7261 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     7321 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     7381 acag
//
