LOCUS       sequence-489004-        6789 bp    DNA     circular SYN 09-OCT-2025
DEFINITION  Cre recombinase-dependent expression of DsRed, i.e. DsRed is only 
            expressed in the presence of Cre due to a transcriptional stop in 
            between the loxP sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6789)
  AUTHORS   Matsuda T, Cepko CL
  TITLE     Controlled expression of transgenes introduced by in vivo 
            electroporation.
  JOURNAL   Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):1027-32. doi: 
            10.1073/pnas.0610155104
  PUBMED    17209010
REFERENCE   2  (bases 1 to 6789)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 9, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1073/pnas"; 
            journalName: "Proc Natl Acad Sci U S A"; date: "2007-01-16- 16"; 
            volume: "104"; issue: "3"; pages: "1027-32"
COMMENT     Sequence Label: pCALNL-DsRed
FEATURES             Location/Qualifiers
     source          1..6789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(1753..1786)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1836..2630
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1890..1909)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2500..2519
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2810..2944
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(2958..2991)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      3063..3072
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3069..3746
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /label=DsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(3249..3269)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     3661..3684
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3831..3850)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3896..3951
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3950..3969)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4312..4328)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4312..4328)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4325..4347)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4336..4352
                     /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(4360..4366,4367..4384,4385..4390))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4405..4426
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      4511..4645
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4665..4799
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4867..4884)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5038..5626)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5118..5137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5797..6588,6589..6657))
                     /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     6420..6439
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6658..6762)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tgattaattc
     1741 gagcgaacgc gtataacttc gtatagcata cattatacga agttatctcg agtcggattt
     1801 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     1861 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     1921 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     1981 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     2041 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     2101 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     2161 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     2221 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     2281 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     2341 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     2401 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     2461 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     2521 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     2581 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     2641 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     2701 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     2761 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccatcgata acttgtttat
     2821 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2881 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     2941 gatcaatccg aacgcgtata acttcgtata gcatacatta tacgaagtta tctcgagtcg
     3001 ctcggtacga tttaaattga attctgcagt cgacggtacc gcgggcccgg gatccaccgg
     3061 tcgccaccat ggcctcctcc gagaacgtca tcaccgagtt catgcgcttc aaggtgcgca
     3121 tggagggcac cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
     3181 acgagggcca caacaccgtg aagctgaagg tgaccaaggg cggccccctg cccttcgcct
     3241 gggacatcct gtccccccag ttccagtacg gctccaaggt gtacgtgaag caccccgccg
     3301 acatccccga ctacaagaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga
     3361 acttcgagga cggcggcgtg gcgaccgtga cccaggactc ctccctgcag gacggctgct
     3421 tcatctacaa ggtgaagttc atcggcgtga acttcccctc cgacggcccc gtgatgcaga
     3481 agaagaccat gggctgggag gcctccaccg agcgcctgta cccccgcgac ggcgtgctga
     3541 agggcgagac ccacaaggcc ctgaagctga aggacggcgg ccactacctg gtggagttca
     3601 agtccatcta catggccaag aagcccgtgc agctgcccgg ctactactac gtggacgcca
     3661 agctggacat cacctcccac aacgaggact acaccatcgt ggagcagtac gagcgcaccg
     3721 agggccgcca ccacctgttc ctgtagcggc cgcactcctc aggtgcaggc tgcctatcag
     3781 aaggtggtgg ctggtgtggc caatgccctg gctcacaaat accactgaga tctttttccc
     3841 tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa
     3901 aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg
     3961 acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca
     4021 acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg tcatcagtat
     4081 atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag
     4141 atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta
     4201 catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct
     4261 cttctcttat ggagatccct cgacctgcag cccaagcttg gcgtaatcat ggtcatagct
     4321 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4381 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4441 actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca
     4501 gcaaccatag tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4561 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     4621 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctaacttg tttattgcag
     4681 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4741 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcc
     4801 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4861 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4921 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4981 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5041 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5101 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5161 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5221 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5281 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5341 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5401 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5461 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5521 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5581 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5641 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5701 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5761 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5821 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5881 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5941 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6001 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6061 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6121 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6181 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6241 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6301 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6361 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6421 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6481 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6541 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6601 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6661 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6721 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6781 gccacctgg
//
