LOCUS       sequence-122974-        7948 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector for homologous recombination to insert a 2A-mCherry-NLS
            cassette in place of the stop codon of myogenin in the mouse 
            genome..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7948)
  AUTHORS   Gersbach
  TITLE     TMG reporters
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7948)
  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: 
            "Unpublished"
COMMENT     Sequence Label: Myog'-2A-mCherryNLS-PGK-Puro
FEATURES             Location/Qualifiers
     source          1..7948
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             964..1020
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1021..1728
                     /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(1166..1184)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1434..1453
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1729..1749
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1756..1776
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    1821..1854
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1915..2411
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1950..1973)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2344..2363
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2451..3050
                     /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(2451..2470)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2947..2967
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    3607..3640
                     /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(4381..4400)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4382..4400)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4407..4424)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4407..4423)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4416..4438)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4564..4992
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4651..4670)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4861..4882
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             5336..6130
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5390..5409)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6000..6019
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(6148..6216,6217..7008)
                     /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(6366..6385)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7179..7767
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7668..7687
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7921..7938
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
      241 gtaccgagct cggatccgtg cccagtgaat gcaactccca cagcgcctcc tgcagtccgg
      301 agtggggcaa tgcactggag ttcggtccca acccaggagg taagtgaatc ctgaccctgg
      361 taacatggct caaatccctt agtccctacc taggctgggc ttgtaccctt accataagcc
      421 ctgtcccaat cacccttcag cagcttttct aggaacttgc tgacctgagg gccccaaatg
      481 gtgggctgga gaccctgtgc ctcccggtag ccttgaccag ctaaagagta gtcagagcct
      541 ccagcaactt ccctgctggt ctctgacctc agagagaaga gcctgctcct gccccagggg
      601 tccctgggga tcactcagtc agtgttgtaa aactgtgccc tgtgccctag gatatcctgg
      661 cttcacctag ggatagccac ttttaaaacc ccacaagcac acacacagca tggggagtac
      721 tagtgtcctg ggggcgggga acactcctga gtcttcctta cccattttgt cctggaaaat
      781 ctgtgtcaag ggtcccatac cagttgccca ggaaacattc tcccactttt tcttgcagat
      841 catttgctcg cggctgaccc tacagacgcc cacaatctgc actcccttac gtccatcgtc
      901 gacagcatca cggtggagga tatgtctgtt gccttcccag acgaaaccat gcccaacgct
      961 agcgccacta acttctccct gttgaaacaa gcaggggatg tcgaagagaa tcccgggcca
     1021 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     1081 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     1141 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     1201 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     1261 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     1321 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     1381 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     1441 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     1501 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     1561 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     1621 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     1681 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagcc caagaagaag
     1741 aggaaggtgg gtggccctaa gaaaaagaga aaggtgtgac gtacaagtaa gaattccgat
     1801 catattcaat aacccttaat ataacttcgt ataatgtatg ctatacgaag ttattaggtc
     1861 tgaagaggag tttacgtcca gccaagctta ggatctcgac ctcgaaattc taccgggtag
     1921 gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg ctggcacttg
     1981 gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta gcgccaaccg
     2041 gctccgttct ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc
     2101 ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact
     2161 agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc
     2221 ggccaatagc agctttgctc cttcgctttc tgggctcagc agctgggaag ggtgggtccg
     2281 ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc
     2341 cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc
     2401 cgggcctttc gacctgcatc catctagatc tcgagcagct gaagcttacc atgaccgagt
     2461 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     2521 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     2581 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     2641 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     2701 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     2761 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     2821 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     2881 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     2941 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     3001 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc
     3061 acgacccgca gcgcccgacc gaaaggagcg cacgacccca tgcatcgatg atatcagatc
     3121 cccgggatgc agaaattgat gatctattaa acaataaaga tgtccactaa aatggaagtt
     3181 tttcctgtca tactttgtta agaagggtga gaacagagta cctacatttt gaatggaagg
     3241 attggagcta cgggggtggg ggtggggtgg gattagataa atgcctgctc tttactgaag
     3301 gctctttact attgctttat gataatgttt catagttgga tatcataatt taaacaagca
     3361 aaaccaaatt aagggccagc tcattcctcc cactcatgat ctatagatct atagatctct
     3421 cgtgggatca ttgtttttct cttgattccc actttgtggt tctaagtact gtggtttcca
     3481 aatgtgtcag tttcatagcc tgaagaacga gatcagcagc ctctgttcca catacacttc
     3541 attctcagta ttgttttgcc aagttctaat tccatcagaa gctggtcgag atccggaacc
     3601 cttaatataa cttcgtataa tgtatgctat acgaagttat taggtccctc gaagaggttc
     3661 actaggcgcg ccgattgtct gtcaggctgg gtgtgcatgt gagcccccaa gttggtgtca
     3721 aaagccatca cttctgtagc agggggcttt taagtggggc tgtcctgatg tccagaaaac
     3781 agccctgggc tgccacaagc cagactcccc actccccatt cacataaggc taacacccag
     3841 cccagcgagg gaatttagct gactccttaa agcagagagc atcctcttct gaggagagaa
     3901 agatggagtc cagagagccc ccttgttaat gtccctcagt ggggcaaact caggagcttc
     3961 ttttttgttt atcataatat gcctcgaatt ccacccccca cccccaaaat gaaaccgttt
     4021 gagagacatg agtgccctga cctggacaag tgtgcacatc tgttctagtc tcttcctgaa
     4081 gccagtggct gggctgggcc tgccctgagt tgagagagaa gggggaggag ctatccggtt
     4141 ccaaagcctc tgggggccaa gcatttgcag tggatcttgg gaaccttcca gtgctttgtg
     4201 tattgtttat tgttttgtgt gttgtttgta aagctgccgt ctgaccaagg tctcctgtgc
     4261 tgatgatacc gggaacaggc agggaagggg gtgggggctc ttggggtgac ttcttttgtt
     4321 aactaagcat tgtgtggttt tgcgcggccg ctcgagcatg catctagagg gcccaattcg
     4381 ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa
     4441 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     4501 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     4561 tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4621 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4681 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4741 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     4801 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4861 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     4921 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     4981 ttaacgcgaa ttttaacaaa attcagggcg caagggctgc taaaggaagc ggaacacgta
     5041 gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg
     5101 gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg
     5161 atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc
     5221 ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat
     5281 ctgatggcgc aggggatcaa gatctgatca agagacagga tgaggatcgt ttcgcatgat
     5341 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     5401 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     5461 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     5521 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     5581 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     5641 cctgtcatcc caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     5701 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     5761 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     5821 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     5881 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     5941 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     6001 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     6061 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     6121 gttcttctga attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     6181 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     6241 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     6301 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     6361 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     6421 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     6481 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     6541 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     6601 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6661 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6721 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6781 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6841 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6901 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6961 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     7021 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     7081 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     7141 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     7201 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     7261 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     7321 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     7381 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     7441 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     7501 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     7561 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     7621 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     7681 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7741 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7801 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     7861 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     7921 agcgagtcag tgagcgagga agcggaag
//
