LOCUS       Exported                4446 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  knock in bait for Axolotl Tyrosinase locus.
ACCESSION   .
VERSION     .
KEYWORDS    pGEMT-Tyrosinase-bait-T2a-Cherry-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4446)
  AUTHORS   Fei JF, Schuez M, Tazaki A, Taniguchi Y, Roensch K, Tanaka EM
  TITLE     CRISPR-mediated genomic deletion of Sox2 in the axolotl shows a 
            requirement in spinal cord neural stem cell amplification during 
            tail regeneration.
  JOURNAL   Stem Cell Reports. 2014 Sep 9;3(3):444-59. doi: 
            10.1016/j.stemcr.2014.06.018. Epub 2014 Aug 7.
  PUBMED    25241743
REFERENCE   2  (bases 1 to 4446)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4446
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     11..33
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     25..42
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     26..42
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        49..66
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             312..365
                     /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             366..1076
                     /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(511..529)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     779..798
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1186..1205)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1251..1306
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1305..1324)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     promoter        complement(1636..1654)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1637..1654)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1672..1688)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1672..1688)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1685..1707)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1696..1712
                     /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(1720..1750)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1765..1786
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1903..1920)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2074..2662)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2154..2173)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2833..3693)
                     /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     3456..3475
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3694..3798)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3876..4331)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4013..4034)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4225..4244
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca
       61 ctatagacgc gtagaacatc gacttcgcgc acgaggcgcc cgccttcctt ccctggcacc
      121 ggctcttcct gctgctctgg gagcgggaga tccagaaggt gaccggcgac caggacttca
      181 ctatccccta ctgggactgg agggacgccc agggctgcgc cgtctgcacc gaccagctga
      241 tgggcgcccg gcaccccacc gtggccaact tactcagccc gggctccttc ttcgcttctt
      301 ggcaggcatg cgagggcaga ggaagtcttc taacatgcgg tgacgtggag gagaatcccg
      361 gccctatggt gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct
      421 tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg
      481 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
      541 tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga
      601 agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg
      661 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc
      721 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc
      781 ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg
      841 acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg
      901 acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca
      961 acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     1021 acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtaagaat
     1081 tctgcagtcg agtcatcgcg attaattcac tcctcaggtg caggctgcct atcagaaggt
     1141 ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt ttccctctgc
     1201 caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa
     1261 atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaaggacata
     1321 tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt tggcaacata
     1381 tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc agtatatgaa
     1441 acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag gttagatttt
     1501 ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt ccttacatgt
     1561 tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg tccctcttct
     1621 cttatgaaga tctattctat agtgtcacct aaatagcttg gcgtaatcat ggtcatagct
     1681 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     1741 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     1801 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     1861 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     1921 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     1981 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2041 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2101 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2161 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2221 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2281 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2341 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2401 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2461 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     2521 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     2581 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     2641 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     2701 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     2761 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     2821 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     2881 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     2941 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3001 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3061 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3121 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3181 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3241 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3301 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3361 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3421 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3481 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     3541 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     3601 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     3661 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     3721 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     3781 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgatgcgg tgtgaaatac
     3841 cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta atattttgtt
     3901 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     3961 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     4021 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     4081 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     4141 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     4201 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     4261 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     4321 acagggcgcg tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     4381 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     4441 gtaacg
//
