LOCUS       sequence-340327-        8416 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encoding dog Nrg1 before Cre-mediated recombination..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8416)
  AUTHORS   Lin S, Hirayama D, Maryu G, Matsuda K, Hino N, Deguchi E, Aoki K, 
            Iwamoto R, Terai K, Matsuda M
  TITLE     Redundant roles of EGFR ligands in the ERK activation waves during 
            collective cell migration.
  JOURNAL   Life Sci Alliance. 2021 Oct 19;5(1). pii: 5/1/e202101206. doi: 
            10.26508/lsa.202101206. Print 2022 Jan.
  PUBMED    34667080
REFERENCE   2  (bases 1 to 8416)
  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: "Life Sci 
            Alliance."; date: "2021-10-19"; volume: "5(1). pii: 5/1/e202101206. 
            doi"; pages: " 10.26508/lsa.202101206. Print 2022 Jan"
COMMENT     Sequence Label: pPBpuro-UbC-loxP-canisNrg1
FEATURES             Location/Qualifiers
     source          1..8416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        138..1349
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     1186..1205
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     protein_bind    complement(1356..1389)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3417..3966
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3561..3578)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3788..3807
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3969..4568
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3969..3988)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4465..4485
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4595..4612)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4601..4825
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(4851..4884)
                     /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(4911..4927)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4935..4951
                     /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(4959..4965,4966..4983,4984..4989))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5004..5025
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   5259..5321
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5362..5382)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5362..5380)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5401..5417)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5425..5441
                     /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(5449..5455,5456..5473,5474..5479))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5494..5515
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5632..5649)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5803..6391)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5883..5902)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6562..7353,7354..7422))
                     /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     7185..7204
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7423..7527)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7553..8008)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7690..7711)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7902..7921
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8135..8157
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8149..8166
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8150..8166
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8176..8195
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8176..8194
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   8224..8258
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 gcatgattat cttttacgtg acttttaaga tttaactcat acgataatta tattgttatt
       61 tcatgttcta cttacgtgat aacttattat atatatattt tcttgttata gatatcaact
      121 agaatgctag cctcgagggc ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct
      181 cctcacggcg agcgctgcca cgtcagacga agggcgcagc gagcgtcctg atccttccgc
      241 ccggacgctc aggacagcgg cccgctgctc ataagactcg gccttagaac cccagtatca
      301 gcagaaggac attttaggac gggacttggg tgactctagg gcactggttt tctttccaga
      361 gagcggaaca ggcgaggaaa agtagtccct tctcggcgat tctgcggagg gatctccgtg
      421 gggcggtgaa cgccgatgat tatataagga cgcgccgggt gtggcacagc tagttccgtc
      481 gcagccggga tttgggtcgc agttcttgtt tgtggatcgc tgtgatcgtc acttggtgag
      541 tagcgggctg ctgggctggc cggggctttc gtggccgccg ggccgctcgg tgggacggag
      601 gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc gagcaaggtt gccctgaact
      661 gggggttggg gggagcgcag caaaatggcg gctgttcccg agtcttgaat ggaagacgct
      721 tgtgaggcgg gctgtgaggt cgttgaaaca aggtgggggg catggtgggc ggcaagaacc
      781 caaggtcttg aggccttcgc taatgcggga aagctcttat tcgggtgaga tgggctgggg
      841 caccatctgg ggaccctgac gtgaagtttg tcactgactg gagaactcgg tttgtcgtct
      901 gttgcggggg cggcagttat ggcggtgccg ttgggcagtg cacccgtacc tttgggagcg
      961 cgcgccctcg tcgtgtcgtg acgtcacccg ttctgttggc ttataatgca gggtggggcc
     1021 acctgccggt aggtgtgcgg taggcttttc tccgtcgcag gacgcagggt tcgggcctag
     1081 ggtaggctct cctgaatcga caggcgccgg acctctggtg aggggaggga taagtgaggc
     1141 gtcagtttct ctggtcggtt ttatgtacct atcttcttaa gtagctgaag ctccggtttt
     1201 gaactatgcg ctcggggttg gcgagtgtgt tttgtgaagt tttttaggca ccttttgaaa
     1261 tgtaatcatt tgggtcaata tgtaattttc agtgttagac tagtaaattg tccgctaaat
     1321 tctggccgtt tttggctttt ttgttagacg gatccataac ttcgtatagc atacattata
     1381 cgaagttatg aattcaccat ggaacccgac gccaacagca gcagtagagc ccctgctgcc
     1441 tttcgggcta gcttcccacc tctggaaacc ggccggaacc tgaagaaaga ggtgtccaga
     1501 gtcctgtgca agagatgcgc cctgcctcct cggctgaaag agatgagaag ccaagagtct
     1561 gccgccggaa gcaagctggt gctgagatgt gaaaccagca gcgagtacag cagcctgaag
     1621 ttcaagtggt tcaagaacgg caacgagctg aaccggaaga acaagcccca gaacatcaag
     1681 atccagaaga agcccggcaa gagcgagctg agaatcagca aagccagcct ggccgatagc
     1741 ggcgagtaca tgtgtaaagt gacctccaag ctgggcaacg acagcgccag cgccaatatc
     1801 accatcgtgg acagcaacga catcatcacc ggcatgcctg ccagcaccga gagggcttat
     1861 gtgtctagcg agagccccat ccggatcagc gttagcacag aaggcgccaa cacaagcagc
     1921 agcacctcta ccagcaccac cggcacatct cacctggtca agtgcgccga gaaagaaaag
     1981 accttctgcg tgaacggcgg cgagtgcttc atggtcaagg acctgagcaa ccccagccgg
     2041 tatctgtgca agtgtcagcc cggttttacc ggcgccagat gcaccgagaa tgtgcccatg
     2101 aaggtgcaga atcaagagaa ggccgaggaa ctgtaccaga aacgggtgct gaccatcaca
     2161 ggcatctgta tcgccctgct ggtcgtgggc attatgtgcg tggtggccta ctgcaagacc
     2221 aagaagcagc ggaagaagct gcacgaccgg ctgagacaga gcctgagaag cgagagaaac
     2281 aacatggtca atatcgccaa cggaccccac catcctaatc ctccacctga gaacgtgcag
     2341 ctggtcaacc agtacgtgtc caagaacgtg atcagctccg agcacatcgt ggaacgcgag
     2401 gccgagacaa gctttagcac cagccactac accagcacag cccaccacag caccacagtg
     2461 acacagaccc caagccacag ctggtccaat ggccacaccg agtccatcat cagcgagtcc
     2521 cacagcgtga tcatgatgag cagcgtggaa aacagccggc actctagccc tagcggagga
     2581 cctagaggca gactgaatgg cctcggcgga cccagagagt gcaacagctt tctgagacac
     2641 gccagagaga cacccgacag ctacagagat agccctcaca gcgagagata cgtgtccgcc
     2701 atgacaaccc ctgccagaat gagccccgtg gactttcaca cacctagcag ccctaagagc
     2761 cctcctagcg agacatcccc tccagtgtct agcacaaccg tgtccatgcc tagcatggcc
     2821 gtgtcaccct tcgtggaaga ggaaagacct ctgctgctgg tcacccctcc tagactgaga
     2881 gagaagtacg atcaccacag ccagcagttc aacagctacc accacaatcc tgctcacgag
     2941 agcaacagcc tgcctccatc tcctctgaga atcgtcgagg acgaggaata cgagacaacc
     3001 caagagtacg agcccgctca agagcccgtg aagaaactga ccagctccag acgggccaag
     3061 cggaccaagc ctaatggaca cattgccaac cggctggaaa tggactccaa tgcctctgcc
     3121 gagggcacca acagcgaaag cgagacagag gatgagagag tgggcgaaga taccccattc
     3181 ctgggcatcc agaatcctct ggccgcctct cttgaagccg ctcctgcttt tagactggcc
     3241 gacagcagaa caaacccagc cggcagattc agcacccaag aggaactgca ggccagactg
     3301 tctagcgtga tcgccaacca ggatcctatc gccgtctgag cggccgctct agagtcaact
     3361 aacttaagct agcaacggtt tccctctagc gggatcaatt ccgccccccc cccctaacgt
     3421 tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac
     3481 catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag
     3541 cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa
     3601 ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag
     3661 gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga
     3721 tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag
     3781 agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc
     3841 ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag
     3901 gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg
     3961 ataataccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca
     4021 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     4081 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     4141 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     4201 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     4261 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     4321 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     4381 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     4441 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     4501 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     4561 gtgcctgatc gatgatctag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     4621 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4681 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     4741 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     4801 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaacctgcag ataacttcgt
     4861 atagcataca ttatacgaag ttattgcagc ccaagcttgg cgtaatcatg gtcatagctg
     4921 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4981 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5041 ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga tcttaaaagt tttgttactt
     5101 tatagaagaa attttgagtt tttgtttttt tttaataaat aaataaacat aaataaattg
     5161 tttgttgaat ttattattag tatgtaagtg taaatataat aaaacttaat atctattcaa
     5221 attaataaat aaacctcgat atacagaccg ataaaacaca tgcgtcaatt ttacgcatga
     5281 ttatctttaa cgtacgtcac aatatgatta tctttctagg gttaatctag tatacgcgtt
     5341 taattaatcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg
     5401 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5461 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5521 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5581 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5641 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5701 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5761 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5821 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5881 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5941 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6001 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6061 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6121 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6181 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6241 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6301 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6361 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6421 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6481 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6541 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6601 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6661 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6721 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6781 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6841 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6901 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6961 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7021 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7081 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7141 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7201 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7261 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7321 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7381 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7441 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7501 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     7561 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     7621 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     7681 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     7741 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     7801 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     7861 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     7921 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     7981 gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg
     8041 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     8101 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     8161 gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc cattctagat
     8221 taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat attgctctct
     8281 ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg ccgcttggag
     8341 ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact ataacgaccg
     8401 cgtgagtcaa aatgac
//
