LOCUS       sequence-192371-        7167 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To clone gene of interest downstream of LoxP3-Mb2-HA-mTFP1-2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7167)
  AUTHORS   Pontes-Quero S, Heredia L, Casquero-Garcia V, Fernandez-Chacon M, 
            Luo W, Hermoso A, Bansal M, Garcia-Gonzalez I, Sanchez-Munoz MS, 
            Perea JR, Galiana-Simal A, Rodriguez-Arabaolaza I, Del Olmo-Cabrera 
            S, Rocha SF, Criado-Rodriguez LM, Giovinazzo G, Benedito R
  TITLE     Dual ifgMosaic: A Versatile Method for Multispectral and 
            Combinatorial Mosaic Gene-Function Analysis.
  JOURNAL   Cell. 2017 Aug 10;170(4):800-814.e18. doi: 
            10.1016/j.cell.2017.07.031.
  PUBMED    28802047
REFERENCE   2  (bases 1 to 7167)
  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: 
            "10.1016/j.cell.2017.07"; journalName: "Cell"; date: "2017-08-10- 
            10"; volume: "170"; issue: "4"; pages: "800-814.e18"
COMMENT     Sequence Label: LoxP3-Mb2-HA-mTFP1-2A (SO157)
FEATURES             Location/Qualifiers
     source          1..7167
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        78..381
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..585
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     536..556
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     630..649
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        630..648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(678..711)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      718..727
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             853..879
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             886..912
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             919..945
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             955..1662
                     /codon_start=1
                     /product="monomeric teal (cyan) fluorescent protein (Ai et 
                     al., 2006)"
                     /label=mTFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFAYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKKTTGWDASTERMYVRDGVLK
                     GDVKHKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYESA
                     VARNSTDGMDELYK"
     CDS             1756..1809
                     /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"
     misc_feature    1862..2450
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1915..1935)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2333..2344)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2584..2705
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2758..2775)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2764..2988
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3034..3462
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3121..3140)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3331..3352
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3471..3491)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3476..3805
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3656..3791
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3718..3737
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3872..4666
                     /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(3926..3945)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4536..4555
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4840..4961
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5010..5026)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5010..5026)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5023..5045)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5034..5050
                     /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(5058..5064,5065..5082,5083..5088))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5103..5124
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5241..5258)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5412..6000)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5492..5511)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6171..6962,6963..7031))
                     /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     6794..6813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7032..7136)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatcctag ttattaatag taatcaatta cggggtcatt agttcatagc
       61 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      121 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      181 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      241 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      301 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      361 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      421 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      481 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      541 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga
      601 gaacccactg cttactggct tatcgaaatt aatacgactc actataggga gacccaagct
      661 ggctagcaat tactagtata acttcgtata gcatacatta tacgaagtta tcggcgcgcc
      721 accatgggtt gctgtttctc caagaccgca gcgaagggag aagccgccgc ggagaggcct
      781 ggggaggcgg ctgtggcctc gtcgccttcc aaagcgaacg gacaggagaa tggccacgtg
      841 aaggtaggat cctacccata cgatgttcct gactatgcgg ccgaatatcc ctatgacgtc
      901 ccggactatg cagccgaata tccatacgac gttccagatt acgctgccaa acttatggtg
      961 agcaagggcg aggagaccac aatgggcgta atcaagcccg acatgaagat caagctgaag
     1021 atggagggca acgtgaatgg ccacgccttc gtgatcgagg gcgagggcga gggcaagccc
     1081 tacgacggca ccaacaccat caacctggag gtgaaggagg gagcccccct gcccttctcc
     1141 tacgacattc tgaccaccgc gttcgcctac ggcaacaggg ccttcaccaa gtaccccgac
     1201 gacatcccca actacttcaa gcagtccttc cccgagggct actcttggga gcgcaccatg
     1261 accttcgagg acaagggcat cgtgaaggtg aagtccgaca tctccatgga ggaggactcc
     1321 ttcatctacg agatacacct caagggcgag aacttccccc ccaacggccc cgtgatgcag
     1381 aagaagacca ccggctggga cgcctccacc gagaggatgt acgtgcgcga cggcgtgctg
     1441 aagggcgacg tcaagcacaa gctgctgctg gagggcggcg gccaccaccg cgttgacttc
     1501 aagaccatct acagggccaa gaaggcggtg aagctgcccg actatcactt tgtggaccac
     1561 cgcatcgaga tcctgaacca cgacaaggac tacaacaagg tgaccgttta cgagagcgcc
     1621 gtggcccgca actccaccga cggcatggac gagctgtata agagtgcacg cgtgagtaag
     1681 ctgaaccctc ctgatgagag tggccccggc tgcatgagct gcaagtgtgt gctctccctg
     1741 tacaagggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat
     1801 cctggcccag aattctgcag atatccagca cagtggcggc cgctcgaggg cgcgccccgc
     1861 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     1921 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     1981 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     2041 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     2101 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     2161 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     2221 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     2281 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     2341 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     2401 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     2461 tcgagactgc tgcaccagat cataatcagc cataccacat ttgtagaggt tttacttgct
     2521 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     2581 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     2641 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     2701 tcttaaggcg tggcggccgc atcgagtcta gagggcccgt ttaaacccgc tgatcagcct
     2761 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     2821 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     2881 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     2941 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     3001 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     3061 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3121 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3181 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3241 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3301 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3361 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3421 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     3481 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3541 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3601 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3661 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3721 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     3781 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     3841 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     3901 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     3961 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     4021 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     4081 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     4141 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     4201 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     4261 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     4321 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     4381 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     4441 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     4501 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     4561 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     4621 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     4681 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     4741 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     4801 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     4861 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4921 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     4981 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     5041 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5101 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5161 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5221 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5281 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5341 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5401 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5461 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5521 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5581 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5641 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5701 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5761 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5821 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5881 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5941 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6001 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6061 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7141 tttccccgaa aagtgccacc tgacgtc
//
