LOCUS       sequence-498225-        5978 bp    DNA     circular SYN 26-JAN-2026
DEFINITION  Donor plasmid for Crispr/Cas9 gene editing of the human MAFA locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5978)
  AUTHORS   Malka-Gibor E, Oliver KM, Chen S, Garcia-Diaz A, Williams D, Henry 
            JA, Akcan BT, Corneo B, de Nooij JC
  TITLE     Derivation and analysis of human somatic sensory neuron subtypes 
            facilitated through fluorescent hPSC reporters.
  JOURNAL   Stem Cell Reports. 2026 Jan 2:102753. doi: 
            10.1016/j.stemcr.2025.102753.
  PUBMED    41483810
REFERENCE   2  (bases 1 to 5978)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 26, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Stem Cell 
            Reports. 2026 Jan 2:102753. doi: 10.1016/j.stemcr.2025.102753."
COMMENT     Sequence Label: pDonor MAFA-P2A-tdTomato
FEATURES             Location/Qualifiers
     source          1..5978
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    713..794
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(750..769)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     804..823
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             1065..1088
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     regulatory      1085..1094
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1533..1589
                     /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             1590..3020
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     rep_origin      complement(3597..4052)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3734..3755)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3946..3965
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4079..4183
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4185..4542
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4185..4376
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4208..4228)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4393..4528
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4577..5371
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4577..5371
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4631..4650)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5241..5260
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5559..5578)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5603..5650
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taaacttgtt
      721 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
      781 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cgttaccagc
      841 atcacctcaa ccccgaggcg ctcaacctga cgcccgagga cgcggtggag gcgctcatcg
      901 gcagcggcca ccacggcgcg caccacggcg cgcaccaccc ggcggccgcc gcagcctacg
      961 aggctttccg cggcccgggc ttcgcgggcg gcggcggagc ggacgacatg ggcgccggcc
     1021 accaccacgg cgcgcaccac gccgcccacc accaccacgc cgcccaccac caccaccacc
     1081 accaccatgg cggcgcggga cacggcggtg gcgcgggcca ccacgtgcgc ctggaggagc
     1141 gcttctccga cgaccagctg gtgtccatgt cggtgcgcga gctgaaccgg cagctccgcg
     1201 gcttcagcaa ggaggaggtc atccggctca agcagaagcg gcgcacgctc aagaaccgcg
     1261 gctacgcgca gtcctgccgc ttcaagcggg tgcagcagcg gcacattctg gagagcgaga
     1321 agtgccaact ccagagccag gtggagcagc tgaagctgga ggtggggcgc ctggccaaag
     1381 agcgggacct gtacaaggag aaatacgaga agctggcggg ccggggcggc cccgggagcg
     1441 cgggcggggc cggtttcccg cgggagcctt cgccgccgca ggccggtccc ggcggggcca
     1501 agggcacggc agacttcttc ctgggatccg gagccacgaa cttctctctg ttaaagcaag
     1561 caggagacgt ggaagaaaac cccggtccta tggtgagcaa gggcgaggag gtcatcaaag
     1621 agttcatgcg cttcaaggtg cgcatggagg gctccatgaa cggccacgag ttcgagatcg
     1681 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     1741 agggcggccc cctgcccttc gcctgggaca tcctgtcccc ccagttcatg tacggctcca
     1801 aggcgtacgt gaagcacccc gccgacatcc ccgattacaa gaagctgtcc ttccccgagg
     1861 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg tctggtgacc gtgacccagg
     1921 actcctccct gcaggacggc acgctgatct acaaggtgaa gatgcgcggc accaacttcc
     1981 cccccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc accgagcgcc
     2041 tgtacccccg cgacggcgtg ctgaagggcg agatccacca ggccctgaag ctgaaggacg
     2101 gcggccacta cctggtggag ttcaagacca tctacatggc caagaagccc gtgcaactgc
     2161 ccggctacta ctacgtggac accaagctgg acatcacctc ccacaacgag gactacacca
     2221 tcgtggaaca gtacgagcgc tccgagggcc gccaccacct gttcctgggg catggcaccg
     2281 gcagcaccgg cagcggcagc tccggcaccg cctcctccga ggacaacaac atggccgtca
     2341 tcaaagagtt catgcgcttc aaggtgcgca tggagggctc catgaacggc cacgagttcg
     2401 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
     2461 tgaccaaggg cggccccctg cccttcgcct gggacatcct gtccccccag ttcatgtacg
     2521 gctccaaggc gtacgtgaag caccccgccg acatccccga ttacaagaag ctgtccttcc
     2581 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggtctg gtgaccgtga
     2641 cccaggactc ctccctgcag gacggcacgc tgatctacaa ggtgaagatg cgcggcacca
     2701 acttcccccc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctccaccg
     2761 agcgcctgta cccccgcgac ggcgtgctga agggcgagat ccaccaggcc ctgaagctga
     2821 aggacggcgg ccactacctg gtggagttca agaccatcta catggccaag aagcccgtgc
     2881 aactgcccgg ctactactac gtggacacca agctggacat cacctcccac aacgaggact
     2941 acaccatcgt ggaacagtac gagcgctccg agggccgcca ccacctgttc ctgtacggca
     3001 tggacgagct gtacaagtag gcgccggacc ccgagcccgc gccgccgtcg ccggggacaa
     3061 gttcgcgcag gcctctcggg gcctcggctc ggactccgcg gtacaggacg tggacaccag
     3121 gcccggcccg gccgtgctgg ccccgctgcc aagtctgcgg gcgcggggct ggaggcccct
     3181 tcgctcccgg tccccgttcg cgcgcgtcgg cccgggtcgc cgtcctgagg ttgagcggag
     3241 aacggtgatt tctaaggaaa cttgagccag gtctaacttc tttccaagcg tccgcttgta
     3301 catacgttga acgtggttct ccgttcccac cttcgccctg ccagcctaga gggaccgcgc
     3361 tgccgtccct tcccgggtgg cccctgcctg cccccgccct ccttcgttct cttctcagcc
     3421 tccctttcct tgcctttttt aacttcccct ccccgtttta aaatcggtct tattttcgaa
     3481 gtatttataa ttattatgct tggtgattag aaaagaaaac cttggaggaa gccccttctt
     3541 tccccagccg gggtccgccc tcagtcgcga gtcacagcat gagtcgctcg aggcgtaaat
     3601 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     3661 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     3721 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     3781 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     3841 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     3901 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     3961 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     4021 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     4081 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4141 ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac
     4201 cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     4261 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     4321 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     4381 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4441 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     4501 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
     4561 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     4621 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4681 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4741 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4801 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4861 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4921 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4981 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     5041 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     5101 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     5161 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     5221 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     5281 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     5341 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     5401 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     5461 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     5521 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     5581 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     5641 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     5701 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     5761 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     5821 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5881 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5941 tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
