LOCUS       sequence-218272-        6439 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyFusion vector for targeting endogeneous genes with mAID-mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6439)
  AUTHORS   Gu B, Posfai E, Rossant J
  TITLE     Efficient generation of targeted large insertions by microinjection 
            into two-cell-stage mouse embryos.
  JOURNAL   Nat Biotechnol. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166.
  PUBMED    29889212
REFERENCE   2  (bases 1 to 6439)
  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 
            Biotechnol. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166."
COMMENT     Sequence Label: EasyFusion mAID-mCherry with Neo
FEATURES             Location/Qualifiers
     source          1..6439
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(21..476)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        503..607
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(608..676,677..1468)
                     /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(826..845)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1639..2227
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2128..2147
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2381..2398
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2515..2536
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2551..2556,2557..2574,2575..2581)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2589..2605
                     /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     2594..2616
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2613..2629
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2613..2629
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2650..2668
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2697..2713
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2698..2714
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2765..2896
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Morawska and Ulrich, 
                     2013)"
                     /label=AID*
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="PKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEAAAFVK"
     CDS             2957..3667
                     /codon_start=1
                     /product="derivative of the monomeric red fluorescent 
                     protein mCherry"
                     /label=mCherry2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFNWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQCRTMGWEASTERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDILSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3102..3120)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    3688..3721
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     3775..3796
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3959..4080
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3996..4015)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4050..4069
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4087..4542)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        4569..4673
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4675..5032
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4675..4866
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4698..4718)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4883..5018
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5067..5861
                     /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             5067..5861
                     /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(5121..5140)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5731..5750
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6049..6068)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(6160..6178)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    6209..6242
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(6274..6293)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6275..6293)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6303..6320)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6303..6319)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6312..6334)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gcagcctgaa tggcgaatgg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
       61 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
      121 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
      181 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
      241 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
      301 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
      361 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
      421 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
      481 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
      541 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
      601 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
      661 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
      721 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
      781 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
      841 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
      901 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
      961 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     1021 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     1081 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     1141 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     1201 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     1261 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     1321 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     1381 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     1441 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     1501 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     1561 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     1621 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     1681 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1741 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     1801 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1861 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     1921 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     1981 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     2041 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     2101 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     2161 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     2221 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     2281 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     2341 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     2401 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     2461 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     2521 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     2581 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
     2641 caagcgcgca attaaccctc actaaaggga acaaaagctg ggtaccgggc cccccctcga
     2701 ggtcgacggt atcgataagc ttgatatcga attcggtagc ggcagcggta gcaagagtgc
     2761 ttgtcctaaa gatccagcca aacctccggc caaggcacaa gttgtgggat ggccaccggt
     2821 gagatcatac cggaagaacg tgatggtttc ctgccaaaaa tcaagcggtg gcccggaggc
     2881 ggcggcgttc gtgaaggtat caatggacgg agcaccgtac ttgaggaaaa tcgatttgag
     2941 gatgtataaa gctagcatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga
     3001 gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga
     3061 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
     3121 gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa
     3181 ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg
     3241 cttcaattgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga
     3301 ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc
     3361 ctccgacggc cccgtaatgc agtgtcgtac catgggctgg gaggcctcca ctgagcggat
     3421 gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg
     3481 cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc
     3541 cggcgcctac aacgtcgaca tcaagttgga catcctttcc cacaacgagg actacaccat
     3601 cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta
     3661 caagtaaact agttctagag cggccgcata acttcgtata atgtatgcta tacgaagtta
     3721 ttacctgagc catcagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt
     3781 cctgctggag ttcgtgaccg ccgccgggat tacacatggc atggacgagc tgtacaagta
     3841 atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     3901 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     3961 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4021 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4081 acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     4141 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     4201 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     4261 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     4321 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     4381 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     4441 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     4501 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     4561 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4621 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg
     4681 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     4741 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     4801 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     4861 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     4921 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     4981 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc
     5041 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     5101 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     5161 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     5221 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     5281 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     5341 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     5401 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     5461 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     5521 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     5581 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     5641 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     5701 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     5761 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     5821 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     5881 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     5941 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     6001 cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa
     6061 ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca
     6121 cggtgttggg tcgttggatc cggtacccag cttttgttcc ctttagtgag ggttaattgc
     6181 gcgcttggcg taatcacggt catagctgat aacttcgtat aatgtatgct atacgaagtt
     6241 atgcggccgc caccgcggtg gagctccaat tcgccctata gtgagtcgta ttacgcgcgc
     6301 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     6361 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     6421 cgcccttccc aacagttgc
//
