LOCUS       sequence-211552-        5575 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  knock in bait for Axolotl Pax7 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5575)
  AUTHORS   Fei JF, Schuez M, Knapp D, Taniguchi Y, Drechsel DN, Tanaka EM
  TITLE     Efficient gene knockin in axolotl and its use to test the role of 
            satellite cells in limb regeneration.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Nov 21;114(47):12501-12506. doi: 
            10.1073/pnas.1706855114. Epub 2017 Oct 31.
  PUBMED    29087939
REFERENCE   2  (bases 1 to 5575)
  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: 
            "10.1073/pnas.1706855114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2017-11-21- 21"; volume: "114"; issue: "47"; pages: 
            "12501-12506"
COMMENT     Sequence Label: pGEMT-Pax7bait-T2a-Cherry-pA
FEATURES             Location/Qualifiers
     source          1..5575
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     17..39
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     31..48
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     32..48
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1447..1500
                     /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"
     CDS             1501..2211
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1646..1664)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1914..1933
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2321..2340)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2386..2441
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2440..2459)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     promoter        complement(2771..2789)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2772..2789)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2807..2823)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2807..2823)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2820..2842)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2831..2847
                     /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(2855..2861,2862..2879,2880..2885))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2900..2921
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3038..3055)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3209..3797)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3289..3308)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3968..4759,4760..4828))
                     /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     4591..4610
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4829..4933)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5011..5466)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5148..5169)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5360..5379
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac
       61 gactcactat agacgcgtat ggcttgtctc cccggagctg tccccagaat gatgcggcca
      121 ggaccagggc agaactaccc acggaccggt ttcccactag aagtgtccac tcctctgggc
      181 cagggcagag tcaaccaact ggggggagtg ttcattaacg ggcgcccgtt gcccaaccac
      241 atccgccaca agatcgtgga gatggcacac cacggcattc ggccctgcgt gatttcacgg
      301 cagctacgtg tgtcccacgg ctgcgtgtcc aagatcctgt gccggtacca ggagacgggg
      361 tccatccggc caggagccat cggaggcagc aagcccaggc aggtggccac tcctgacgtg
      421 gagaagaaga ttgaggaata taagagagag aaccccggga tgttcagctg ggagatcagg
      481 gacaggctcc taaaggatgg acactgcgac aggagcacgg tgccctcagg taaccggctg
      541 gacgaaggtt cagatgttga gtcagagcca gacttgccct tgaaacgcaa gcagcgacgc
      601 agccgcacca ccttcactgc tgagcagctg gaggagttgg agaaggcctt tgagagaacg
      661 cactacccag atatctacac ccgtgaagag ttggcgcaga gaaccaagct caccgaggcg
      721 agagtacagg tgtggttcag caatcgtaga gcaaggtggc gtaaacaggc aggagccaat
      781 caactggcag ctttcaatca tctcctgccc ggcggatttc cacccacagg aatgccaagt
      841 ttgccaccct atcagcttcc agactccggc tatgcaagtg ccctagcaca agacggggga
      901 agcacagtgc acagacctca gcccctgcca ccctcctcca tgcaccaggg tggattgtca
      961 gctgctgatg ccagttcagc ctacggaccc cgacacagct tcaccagcta ttcggacagc
     1021 ttcatgaacc caggcgggcc aactaaccac atgaatccgg tcagcaatgg gctgtctcca
     1081 caggtgatga gcatcctcag caaccctagt ggagtgcagt cccagtccca ggatttctcc
     1141 atctcccctc tgcatggtgg cttggacact tcgaactcca tctcggcaag ctgcagccag
     1201 cggagcgact ccatcaagtc agtcgatagc ctcgccacct cgcagtccta ctgtcctccg
     1261 acctacagca ccaccggcta cagcgtggat cctgtggcca gctaccaata tgggcaatac
     1321 gggcaaactg ctgtagacta cttggctaag aacgtgagct tgtcgacaca gaggaggatg
     1381 aagctcgggg accactcagc cgtgttgggt ttgcttcctg tggagacggg acaagcatac
     1441 gcatgcgagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct
     1501 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     1561 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     1621 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     1681 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     1741 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     1801 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     1861 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     1921 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     1981 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     2041 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     2101 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     2161 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta agaattctgc
     2221 agtcgagtca tcgcgattaa ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg
     2281 ctggtgtggc caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa
     2341 attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta
     2401 ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga
     2461 gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc
     2521 atatgctggc tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc
     2581 cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta
     2641 tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac
     2701 tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat
     2761 gaagatctat tctatagtgt cacctaaata gcttggcgta atcatggtca tagctgtttc
     2821 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     2881 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     2941 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3001 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3061 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3121 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3181 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3241 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3301 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3361 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     3421 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3481 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3541 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3601 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     3661 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3721 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     3781 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     3841 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     3901 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3961 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4021 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4081 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4141 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4201 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4261 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4321 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4381 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4441 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4501 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4561 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4621 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     4681 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     4741 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     4801 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     4861 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     4921 taggggttcc gcgcacattt ccccgaaaag tgccacctga tgcggtgtga aataccgcac
     4981 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat
     5041 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     5101 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     5161 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     5221 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     5281 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     5341 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     5401 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     5461 gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     5521 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgg
//
