LOCUS       sequence-349416-        5639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP::mAID donor backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5639)
  AUTHORS   Negishi T, Kitagawa S, Horii N, Tanaka Y, Haruta N, Sugimoto A, Sawa
            H, Hayashi KI, Harata M, Kanemaki MT
  TITLE     The auxin-inducible degron 2 (AID2) system enables controlled 
            protein knockdown during embryogenesis and development in 
            Caenorhabditis elegans.
  JOURNAL   Genetics. 2021 Dec 2. pii: 6448232. doi: 10.1093/genetics/iyab218.
  PUBMED    34865044
REFERENCE   2  (bases 1 to 5639)
  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: "Genetics. 
            2021 Dec 2. pii: 6448232. doi: 10.1093/genetics/iyab218."
COMMENT     Sequence Label: pGFPmAID
FEATURES             Location/Qualifiers
     source          1..5639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(33..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     355..374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(784..1089)
                     /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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(1513..1541)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2289..2309
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             complement(2381..2398)
                     /codon_start=1
                     /product="HQHQHQ metal affinity tag"
                     /label=HQ tag
                     /note="The HQ tag binds to nickel and other metal ions much
                     like the 6xHis tag, but may allow elution at lower 
                     concentrations of imidazole (Godat et al., 2008)."
                     /translation="HQHQHQ"
     CDS             2404..2607
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             complement(2749..3054)
                     /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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     promoter        complement(3454..3472)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3493..3509)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3493..3509)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3506..3528)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3517..3533
                     /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(3541..3547,3548..3565,3566..3571))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3586..3607
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3724..3741)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3895..4483)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3975..3994)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4654..5445,5446..5514))
                     /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     5277..5296
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5515..5619)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggactagtg
      661 tacgggccca gtcgttcggc ttcatctgga ttttcagcct ctatacttac taaacgtgat
      721 aaagtttctg taatttctac tgtatcgacc tgcagactgg ctgtgtataa gggagcctga
      781 catttatatt ccccagaaca tcaggttaat ggcgtttttg atgtcatttt cgcggtggct
      841 gagatcagcc acttcttccc cgataacgga gaccggcaca ctggccatat cggtggtcat
      901 catgcgccag ctttcatccc cgatatgcac caccgggtaa agttcacggg agactttatc
      961 tgacagcaga cgtgcactgg ccagggggat caccatccgt cgcccgggcg tgtcaataat
     1021 atcactctgt acatccacaa acagacgata acggctctct cttttatagg tgtaaacctt
     1081 aaactgcatt tcaccagccc ctgttctcgt cagcaaaaga gccgttcatt tcaataaacc
     1141 gggcgacctc agccatccct tcctgatttt ccgctttcca gcgttcggca cgcagacgac
     1201 gggcttcatt ctgcatggtt gtgcttacca gaccggagat attgacatca tatatgcctt
     1261 gagcaactga tagctgtcgc tgtcaactgt cactgtaata cgctgcttca tagcatacct
     1321 ctttttgaca tacttcgggt atacatatca gtatatattc ttataccgca aaaatcagcg
     1381 cgcaaatacg catactgtta tctggctttt agtaagccgg atccacgcgg cgtttacgcc
     1441 ccccctgcca ctcatcgcag tactgtgggc cctaaactag taatgagtaa aggagaagaa
     1501 cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa
     1561 ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt
     1621 atttgcacta ctggaaaact acctgttcca tgggtaagtt taaacatata tatactaact
     1681 aaccctgatt atttaaattt tcagccaaca cttgtcacta ctttctgtta tggtgttcaa
     1741 tgcttctcga gatacccaga tcatatgaaa cggcatgact ttttcaagag tgccatgccc
     1801 gaaggttatg tacaggaaag aactatattt ttcaaagatg acgggaacta caagacacgt
     1861 aagtttaaac agttcggtac taactaacca tacatattta aattttcagg tgctgaagtc
     1921 aagtttgaag gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagaa
     1981 gatggaaaca ttcttggaca caaattggaa tacaactata actcacacaa tgtatacatc
     2041 atggcagaca aacaaaagaa tggaatcaaa gttgtaagtt taaacatgat tttactaact
     2101 aactaatctg atttaaattt tcagaacttc aaaattagac acaacattga agatggaagc
     2161 gttcaactag cagaccatta tcaacaaaat actccaattg gcgatggccc tgtcctttta
     2221 ccagacaacc attacctgtc cacacaatct gccctttcga aagatcccaa cgaaaagaga
     2281 gaccacatgg tccttcttga gtttgtaaca gctgctggga ttacacatgg catggatgaa
     2341 ctatacaaat gtctagagga tcgggggccc ggtgccggtg ctggtgctgg tgctggtgcc
     2401 ggtaaagaaa agagtgcttg cccaaaagac cctgctaaac ctcctgccaa ggctcaagtt
     2461 gtaggatggc cacctgttag atcatacaga aagaatgtca tggtttcttg tcaaaagtct
     2521 tcaggtggac cagaagctgc agcctttgtt aaagtatcta tggatggtgc cccatatttg
     2581 agaaagattg acttaaggat gtacaagggt gctggagcag gcgccactag tagttaacgt
     2641 tcggcttcat ctggattttc agcctctata cttactaaac gtgataaagt ttctgtaatt
     2701 tctactgtat cgacctgcag actggctgtg tataagggag cctgacattt atattcccca
     2761 gaacatcagg ttaatggcgt ttttgatgtc attttcgcgg tggctgagat cagccacttc
     2821 ttccccgata acggagaccg gcacactggc catatcggtg gtcatcatgc gccagctttc
     2881 atccccgata tgcaccaccg ggtaaagttc acgggagact ttatctgaca gcagacgtgc
     2941 actggccagg gggatcacca tccgtcgccc gggcgtgtca ataatatcac tctgtacatc
     3001 cacaaacaga cgataacggc tctctctttt ataggtgtaa accttaaact gcatttcacc
     3061 agcccctgtt ctcgtcagca aaagagccgt tcatttcaat aaaccgggcg acctcagcca
     3121 tcccttcctg attttccgct ttccagcgtt cggcacgcag acgacgggct tcattctgca
     3181 tggttgtgct taccagaccg gagatattga catcatatat gccttgagca actgatagct
     3241 gtcgctgtca actgtcactg taatacgctg cttcatagca tacctctttt tgacatactt
     3301 cgggtataca tatcagtata tattcttata ccgcaaaaat cagcgcgcaa atacgcatac
     3361 tgttatctgg cttttagtaa gccggatcca cgcggcgttt acgccccccc tgccactcat
     3421 cgcagtactg tactagttaa ccagcttttg ttccctttag tgagggttaa tttcgagctt
     3481 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     3541 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3601 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3661 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3721 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3781 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3841 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3901 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3961 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4021 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4081 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     4141 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     4201 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     4261 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4321 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4381 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4441 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4501 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4561 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4621 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4681 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4741 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4801 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4861 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4921 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4981 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     5041 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     5101 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     5161 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     5221 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     5281 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     5341 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5401 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5461 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5521 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5581 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgc
//
