LOCUS       sequence-67205-e        4655 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to make an RNA probe for whole-mount in situ 
            hybridization, for in-frame fusion proteins or as a subcloning step 
            for mRFP. Note that the coding sequence for mRFP lacks the start 
            codon..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4655)
  AUTHORS   Clark KJ, Balciunas D, Pogoda HM, Ding Y, Westcot SE, Bedell VM, 
            Greenwood TM, Urban MD, Skuster KJ, Petzold AM, Ni J, Nielsen AL, 
            Patowary A, Scaria V, Sivasubbu S, Xu X, Hammerschmidt M, Ekker SC
  TITLE     In vivo protein trapping produces a functional expression codex of 
            the vertebrate proteome.
  JOURNAL   Nat Methods. 2011 Jun;8(6):506-15. Epub 2011 May 8.
  PUBMED    21552255
REFERENCE   2  (bases 1 to 4655)
  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: "Nat 
            Methods."; date: "2011-06"; volume: "8(6)"; pages: "506-15. Epub 
            2011 May 8"
COMMENT     Sequence Label: pCR4-*mRFP
FEATURES             Location/Qualifiers
     source          1..4655
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             309..983
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     primer_bind     complement(436..454)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     704..723
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     898..921
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1026..1045)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1027..1045)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1052..1069)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1052..1068)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1061..1083)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1206..1508
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1387..1406
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1857..2651
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             1857..2651
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1911..1930)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2521..2540
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     promoter        2750..2854
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2855..2923,2924..3715)
                     /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(3073..3092)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3886..4474
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4375..4394
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4628..4645
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctca
      241 gaattaaccc tcactaaagg gactagtcct gcaggtttaa acgaattcgc ccttaagaat
      301 tcgaaggtgc ctcctccgag gatgtcatca aggagttcat gcgcttcaag gtgcgcatgg
      361 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
      421 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg
      481 acatcctgtc ccctcagttc cagtacggct ccaaggccta cgtgaagcac cccgccgaca
      541 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
      601 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
      661 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
      721 agaccatggg ctgggaggcc tccaccgagc ggatgtaccc cgaggacggc gccctgaagg
      781 gcgagatcaa gatgaggctg aagctgaagg acggcggcca ctacgacgcc gaggtcaaga
      841 ccacctacat ggccaagaag cccgtgcagc tgcccggcgc ctacaagacc gacatcaagc
      901 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgcgccgagg
      961 gccgccactc caccggagcc taagactagt ttaagggcga attcgcggcc gctaaattca
     1021 attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact
     1081 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     1141 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctatacg
     1201 tacggcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg
     1261 tacagagtga tattattgac acgccggggc gacggatggt gatccccctg gccagtgcac
     1321 gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa
     1381 gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag
     1441 tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg
     1501 gaatataaat gtcaggcatg agattatcaa aaaggatctt cacctagatc cttttcacgt
     1561 agaaagccag tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct
     1621 ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc
     1681 gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc
     1741 cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga
     1801 tctgatggcg caggggatca agctctgatc aagagacagg atgaggatcg tttcgcatga
     1861 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     1921 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     1981 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     2041 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     2101 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     2161 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     2221 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     2281 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     2341 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     2401 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     2461 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     2521 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     2581 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     2641 agttcttctg aattattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc
     2701 tgtgcggtat ttcacaccgc atacaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2761 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2821 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     2881 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     2941 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     3001 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     3061 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     3121 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     3181 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     3241 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     3301 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     3361 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     3421 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     3481 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     3541 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     3601 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     3661 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     3721 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     3781 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     3841 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3901 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3961 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4021 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4081 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4141 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4201 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4261 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4321 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4381 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4441 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4501 gttcctgggc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4561 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4621 cgagcgcagc gagtcagtga gcgaggaagc ggaag
//
