LOCUS       sequence-316796-        7328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology-directed recombination donor vector for EGFP reporter 
            knock-in to human PAX7 3' UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7328)
  AUTHORS   Xi H, Young CS, Pyle AD
  TITLE     Generation of PAX7 Reporter Cells to Investigate Skeletal Myogenesis
            from Human Pluripotent Stem Cells.
  JOURNAL   STAR Protoc. 2020 Nov 5;1(3):100158. doi: 
            10.1016/j.xpro.2020.100158. eCollection 2020 Dec 18.
  PUBMED    33377052
REFERENCE   2  (bases 1 to 7328)
  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: "STAR 
            Protoc."; date: "2020-11-5"; volume: "1(3):100158. doi"; pages: " 
            10.1016/j.xpro.2020.100158. eCollection 2020 Dec 18"
COMMENT     Sequence Label: pHX_HDR_hPAX7_IRES-EGFP-PGK-Neo
FEATURES             Location/Qualifiers
     source          1..7328
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(677..1251,1252..1254,1255..1263)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(844..861)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1071..1090
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1264..1266,1267..1269,1270..1983)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1264..1266,1267..1269,1270..1980)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1309..1330)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1570..1589)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1917..1938
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2034..2067
                     /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(2094..2117)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2491..2510
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2572..3375
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2635..2654)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3245..3264
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3407..3424)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3413..3524
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3612..3645
                     /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(4399..4415)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4399..4415)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4447..4466)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4448..4466)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4473..4490)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4473..4489)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4482..4504)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4631..5086
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4718..4737)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4928..4949
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5112..5216
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5217..5285,5286..6077)
                     /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(5435..5454)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6248..6836
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6737..6756
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6990..7007
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7124..7145
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7160..7165,7166..7183,7184..7190)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7198..7214
                     /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     7203..7225
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7222..7238
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7222..7238
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        7259..7277
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     7305..7321
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     7306..7322
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 agaaccaagc tatgctttta tacttccatt ttatacttga tttagctttg ttgttctttt
       61 gccagaggaa catcaaaggg cagagcaaac agagaactag ggggataaga gagaagaaaa
      121 gaagaatgga gagagaggtt tgcccagaag gaaaacgaag agccagattg aaatctgact
      181 ctggcttgag aacaggacgg gtcccagagt ttgaccactg ccagaatcca gtgtttcaag
      241 atggagttgg tgaagcatcc aagattttgg atcctgggcc tcagtgacac tccatcaccc
      301 aactaaaaag atgttctctg ggacctctct caggctgatg ttcttctcat caagctcccg
      361 aatgtgtctc gcactattgg acactttttg ggggtacaca ggtcttctct cctcccctcc
      421 tgcagaggca ccaaaaccaa gaccaaatgg gggttctcta gccgccaact tgaaacgctc
      481 tgagacttct gtgtatttga tgcttcttca agtcagctct gacgtggcca attccctgac
      541 ctgggaaata tttacatagt actctagatc attccttctt cctagtaact gcaactttgt
      601 tgcttctgtg tttgactcac agccactttc ccccatgcgg aagtgagtgg gctcagtgat
      661 gctgttgatc gatccgcccc tctccctccc ccccccctaa cgttactggc cgaagccgct
      721 tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg
      781 gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt
      841 cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg
      901 aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac
      961 ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg
     1021 cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct
     1081 caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg
     1141 atctggggcc tcggtacaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag
     1201 gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca
     1261 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     1321 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     1381 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1441 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     1501 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1561 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1621 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1681 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     1741 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     1801 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     1861 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     1921 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     1981 taaagcggcc gatccactag taacggccgc cagtgtgctg gaattcggct tcaacaactt
     2041 cgtataatgt atgctatacg aagttatcag tacttttccc aaggcagtct ggagcatgcg
     2101 ctttagcagc cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt
     2161 ccacatccac cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt
     2221 ctactcctcc cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt
     2281 gacaaatgga agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat
     2341 ggaagcgggt aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg
     2401 ctcagaggct gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg
     2461 cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg
     2521 ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac ctgcagccaa tatgggatcg
     2581 gccattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     2641 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     2701 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     2761 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     2821 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     2881 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     2941 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3001 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3061 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     3121 ggcgatgatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3181 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     3241 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     3301 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     3361 gacgagttct tctgagggga tcaattctct agagctcgct gatcagcctc gactgtgcct
     3421 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3481 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3541 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3601 aatagcaggc aacaacttcg tataatgtat gctatacgaa gttatcagta caagccgaat
     3661 tctgcagtgt acgtgagggt gtgagtgggg tgtgtgcgtg ggcgtgtggg cgtgaggtgt
     3721 gtgtttaaat ataatcgcac cataatttat tcagctatat ggagtagtag actagaaaga
     3781 gaaactggta tttgctttaa ctacctgctg cttgtaagcg cttcctctgt aactcaggcg
     3841 taactgttat acattaaaag aaattaaaag cattttgaag gtttcgtgta ctcaattgat
     3901 tgtcattatg cagctgctcc aaaagggact ctgaggggtg tccaagaagc ctttggacct
     3961 aatttagagg atcagcgtag aaactgtgtt tctcttgctc cccactgggt cctctagtgc
     4021 caagcagctg cgaggtttct agtgaatgcc taatcaatat ttgttcaatg gatgttgtta
     4081 ctttaagcct cgctctgagc tcatagaaac agcacagtgt caaagaataa ggtgaaactt
     4141 tagttctctt caccagagca aacttttcct gctgctaatc ttagaaccct cagtggctgt
     4201 ctcccattac ataggatttg ggaggtagaa gagagctcag aaacttacat attgcttgga
     4261 agcatatggg gattctcggg caaatttctg gctttgttct ttttagagat ggggtcttgc
     4321 tatgttgccc aggctggagt gcaaggctat tcacaggtgt gatcatcctg tgctatggag
     4381 aattcctgca gcccggggga tccactagtt ctagagcggc cgccaccgcg gtggagctcc
     4441 aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac
     4501 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     4561 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     4621 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4681 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4741 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4801 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     4861 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4921 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     4981 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5041 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     5101 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5161 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5221 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5281 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5341 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5401 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     5461 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     5521 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     5581 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     5641 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     5701 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     5761 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     5821 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     5881 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     5941 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     6001 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     6061 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     6121 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6181 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6241 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6301 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6361 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     6421 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6481 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6541 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6601 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6661 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6721 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6781 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6841 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     6901 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     6961 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     7021 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     7081 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     7141 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     7201 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagctcgaaa
     7261 ttaaccctca ctaaagggaa caaaagctgg taccgggccc cccctcgagg tcgacggtat
     7321 cgataagc
//
