LOCUS       sequence-440615-        8549 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology directed repair template for knocking in mGreenLantern 
            reporter to DAZL..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8549)
  AUTHORS   Pierson Smela M, Kramme CC, Fortuna PRJ, Wolf B, Srikar Kavirayuni 
            V, Adams J, Ma C, Velychko S, Widocki U, Goel S, Chen T, Vincoff S, 
            Dong E, Kohman RE, Kobayashi M, Shioda T, Church GM, Chatterjee P
  TITLE     Rapid Human Oogonia-like Cell Specification via Combinatorial 
            Transcription Factor-Directed Differentiation
  JOURNAL   bioRxiv 2022.07.11.499564
REFERENCE   2  (bases 1 to 8549)
  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: "bioRxiv 
            2022.07.11.499564"
COMMENT     Sequence Label: pHDR2_DAZL-T2A-mGreenLantern-PuroTK
FEATURES             Location/Qualifiers
     source          1..8549
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(891..908)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    897..1121
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    1169..1200
                     /label=rox
                     /bound_moiety="Dre recombinase"
     CDS             2669..3241
                     /codon_start=1
                     /gene="Corynebacterium diphtheriae tox"
                     /product="diphtheria toxin fragment A"
                     /label=DTA
                     /translation="DDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYD
                     DDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKEL
                     GLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEI
                     NFETRGKRGQDAMYEYMAQACAGNRVRR"
     intron          3318..3383
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     rep_origin      complement(3666..4254)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3746..3765)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4425..5216,5217..5285))
                     /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     5048..5067
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5286..5390)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6497..6499,6500..6502,6503..7216)
                     /codon_start=1
                     /product="monomeric GFP derivative that displays 
                     exceptionally high brightness in cells (Campbell et al., 
                     2020)"
                     /label=mGreenLantern
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLGYGVACFARYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIVLKGIDFKEDGNILGHKLEYNFNSHKVYITADKQKNGIK
                     ANFKTRHNVEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLK
                     ERVTAAGITHDMDELYK"
     primer_bind     complement(6542..6563)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    7217..7248
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        7298..7797
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(7333..7356)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     7730..7749
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      7811..7820
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7817..8416
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MGTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     8316..8336
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             join(8423..8549,1..869)
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
ORIGIN
        1 ggcaggtgct gggggcttcc gagacaatcg cgaacatcta caccacacaa caccgcctcg
       61 accagggtga gatatcggcc ggggacgcgg cggtggtaat gacaagcgcc cagataacaa
      121 tgggcatgcc ttatgccgtg accgacgccg ttctggctcc tcatatcggg ggggaggctg
      181 ggagctcaca tgccccgccc ccggccctca ccctcatctt cgaccgccat cccatcgccg
      241 ccctcctgtg ctacccggcc gcgcgatacc ttatgggcag catgaccccc caggccgtgc
      301 tggcgttcgt ggccctcatc ccgccgacct tgcccggcac aaacatcgtg ttgggggccc
      361 ttccggagga cagacacatc gaccgcctgg ccaaacgcca gcgccccggc gagcggcttg
      421 acctggctat gctggccgcg attcgccgcg tttacgggct gcttgccaat acggtgcggt
      481 atctgcaggg cggcgggtcg tggcgggagg attggggaca gctttcgggg acggccgtgc
      541 cgccccaggg tgccgagccc cagagcaacg cgggcccacg accccatatc ggggacacgt
      601 tatttaccct gtttcgggcc cccgagttgc tggcccccaa cggcgacctg tacaacgtgt
      661 ttgcctgggc cttggacgtc ttggccaaac gcctccgtcc catgcacgtc tttatcctgg
      721 attacgacca atcgcccgcc ggctgccggg acgccctgct gcaacttacc tccgggatgg
      781 tccagaccca cgtcaccacc cccggctcca taccgacgat ctgcgacctg gcgcgcacgt
      841 ttgcccggga gatgggggag gctaactgag ctctagagct cgctgatcag cctcgactgt
      901 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
      961 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     1021 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     1081 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     1141 cagctggggc tcgacatact agagtactta actttaaata atgccaatta tttaaagtta
     1201 ggcgcgccga tatctagtgg cttatctagt tacatgggaa gttgctggtt ttgaatatta
     1261 agctaaaagg tttccactat tatagaaatt ctgaattttg gtaaatcaca ctcaaacttt
     1321 gtgtataagt tgtattatta gactctctag ttttatctta aactgttctt cattagatgt
     1381 ttatttagaa actggttctg tgttgaaata tagttgaaag taaaaaaata attgagactg
     1441 aaagaaacta agatttatct gcaaggattt tttaaaaatt ggcattttaa gtgtttaaaa
     1501 gcaaatactg attttcaaaa aaatgttttt aaaaacctat tttgaaaggt cagaattttg
     1561 ttggtctgaa tacaaacatt tcacttctcc aacaagtacc tgtgaacagt acagtattta
     1621 cagtattgag ctttgcattt atgatttctc cagaaattta ccacaaaagc aaaattttta
     1681 aaactgcatt tttaatcagt ggaactcaat atatagttag ctttattgaa gtcttcttat
     1741 ctaaacccag caaaacagat tcaaagcgaa cagtccaatc agtgggtcat atgtttattc
     1801 aaaatatttt atcttttagc tagaatccac acatatatat cctatttgat tagggtagta
     1861 attaggataa ctaaaattct gggcctaatt ttttaaagaa tccaagacaa actaaacttt
     1921 actaggtaca taagcttctc aatgagtcac cattcttctt ttttgtaaaa acttttttct
     1981 ttgaaatgct aaacttggct gtatgtcaaa ttgtgcaaaa tattggtatt aaagaatgct
     2041 gcaacttttt tatgtctctt agaggttaat cagagtatct gaagggaatt gtttttataa
     2101 aaacattgaa atattagtta cttgctataa atagatttag tctgttatat ttccttttgt
     2161 aaagtaaaat atgtccagaa gagtcaaagt agttagtttt ggttatttct aaaccacaaa
     2221 agttgtttaa taagtatatc ttaagaatgt gctagagtta aaagttagca ttgtttctag
     2281 attagctggt gtcttcattt tacattgtga caaacagcta gagcatcaga gcccttttgc
     2341 tataccacag tctttcgttt ccagccacct ccagatcttg tcagtgtggt tttcaatagg
     2401 aagcaaaaag cctctccacc caggcctgga atgtttccac ccaatgtcga gcagtgtggt
     2461 tttgcaagag gaagcaaaaa gcctctccac ccaggcctgg aatgtttcca cccaatgtcg
     2521 agcaaacccc gcccagcgtc ttgtcattgg cgaattcgaa cacgcagatg cagtcggggc
     2581 ggcgcggtcc caggtccact tcgcatatta aggtgacgcg tgtggcctcg aacaccgagc
     2641 gaccctgcag gtcctcgcca tggatcctga tgatgttgtt gattcttcta aatcttttgt
     2701 gatggaaaac ttttcttcgt accacgggac taaacctggt tatgtagatt ccattcaaaa
     2761 aggtatacaa aagccaaaat ctggtacaca aggaaattat gacgatgatt ggaaagggtt
     2821 ttatagtacc gacaataaat acgacgctgc gggatactct gtagataatg aaaacccgct
     2881 ctctggaaaa gctggaggcg tggtcaaagt gacgtatcca ggactgacga aggttctcgc
     2941 actaaaagtg gataatgccg aaactattaa gaaagagtta ggtttaagtc tcactgaacc
     3001 gttgatggag caagtcggaa cggaagagtt tatcaaaagg ttcggtgatg gtgcttcgcg
     3061 tgtagtgctc agccttccct tcgctgaggg gagttctagc gttgaatata ttaataactg
     3121 ggaacaggcg aaagcgttaa gcgtagaact tgagattaat tttgaaaccc gtggaaaacg
     3181 tggccaagat gcgatgtatg agtatatggc tcaagcctgt gcaggaaatc gtgtcaggcg
     3241 atctctttgt gaaggaacct tacttctgtg gtgtgacata attggacaaa ctacctacag
     3301 agatttaaag ctctaaggta aatataaaat ttttaagtgt ataatgtgtt aaactactga
     3361 ttctaattgt ttgtgtattt tagattccaa cctatggaac tgatgaatgg gagcagtggt
     3421 ggaatgcaga tccactagag tcgagggatg cagaaattga tgatctatta aacaataaag
     3481 atgtccacta aaatggaagt ttttcctgtc atactttgtt aagaagggtg agaacagagt
     3541 acctacattt tgaatggaag gattggagct acgggggtgg gggtggggtg ggattagata
     3601 aatgcctgct ctttactgaa ggctctttac tattgcttta tgataatgtt tcatagttgg
     3661 atgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3721 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3781 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3841 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3901 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3961 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4021 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4081 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4141 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4201 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4261 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4321 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4381 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4441 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4501 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4561 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4621 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4681 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4741 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4801 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4861 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4921 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4981 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5041 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5101 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5161 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5221 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5281 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5341 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5401 cgacgaatca gaagttggca tcctgtggtt gtctgacatc tagacctatt ttgattgact
     5461 gcatgttgtt gtattgaatt tgaatgcatg aggtattttt gaatgtattt actatttcca
     5521 tagctaattc cactcttcac tgtcttgatt ctgtagtccc ttcataatct tgtttcctgc
     5581 gtgacctgtg aagatacatg ttggtgacca gttttctaga ttttaactta aataggatat
     5641 cactcttttg acagattagg taacttccag aagccacttt tatttatatg acagtgaaac
     5701 tgaggtccta ggaaaagggc acagttaact ttgtagaaaa taaaatgcta ttatgttatt
     5761 actcatttgc ttagtcccat ttctcctttt aaggcctctc acttttttct ctctgcacat
     5821 ctgtaggcaa catagagtga aaagaaagtt ttgcatgtat ttaaaatttt tctctttctt
     5881 tctaaagaat aatacatctt cacaggttaa tgatatgtct ttaaatgcca aaacttacat
     5941 cttttaacct aaaaacatga aatttcagat tggagagctg ttcacaagcc gtggttccta
     6001 ttagatgcag ttcagtcagt gaaaacagta ttttttagaa ttacattttc taccagctgt
     6061 ctttgggaca ttactgcaaa atcattaatt aagaagtaca taaaatgata ttgagtctaa
     6121 gtcctgttat ttctgagttt aatgggattt cttttttttt aattgatttc ttttttaact
     6181 aaactgttta ataaaactag ccatcctggt atatatgtta tcccagtgtt caagaatgct
     6241 tctcaaaaag aataatcttt tttctcatta tttataatgt ttaaacccaa aacaaatggt
     6301 ttaagttttt gacaactttc agatctatag tagtaatcag aaattttcag taaagtaaaa
     6361 ggactctttc tgtcttttcc aggataaaag agtgcatcac tttagaagaa gtcgggcaat
     6421 gcttaaatct gttggctctg gcgagggcag aggaagtctg ctagcatgcg gtgacgtcga
     6481 ggagaatcct ggcccaatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     6541 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtccgcg gcgagggcga
     6601 gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     6661 cgtgccctgg cccaccctcg tgaccacctt aggctacggc gtggcctgct tcgcccgcta
     6721 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     6781 ggagcgcacc atctctttca aggacgacgg tacctacaag acccgcgccg aggtgaagtt
     6841 cgagggcgac accctggtga accgcatcgt gctgaagggc atcgacttca aggaggacgg
     6901 caacatcctg gggcacaagc tggagtacaa cttcaacagc cacaaggtct atatcacggc
     6961 cgacaagcag aagaacggca tcaaggctaa cttcaagacc cgccacaacg ttgaggacgg
     7021 cggcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     7081 gctgcccgac aaccactacc tgagccatca gtccaaactg agcaaagacc ccaacgagaa
     7141 gcgcgatcac atggtcctga aggagagggt gaccgccgcc gggattacac atgacatgga
     7201 cgagctgtac aagtaataac tttaaataat gccaattatt taaagttaca tatgagaaag
     7261 tataggaact tcagcttgat atcgaattaa ttctaccggg taggggaggc gcttttccca
     7321 aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta cacaagtggc
     7381 ctctgccctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc gttctttggt
     7441 ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc cgccccgcag
     7501 ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct cgtgcagatg
     7561 gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca atagcagctt
     7621 tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg cgggctcagg
     7681 ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg
     7741 cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg cctttcgacc
     7801 tgcagccaac gccaccatgg ggaccgagta caagcccacg gtgcgcctcg ccacccgcga
     7861 cgacgtcccc cgggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     7921 ccacaccgtc gacccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
     7981 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc
     8041 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     8101 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     8161 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca
     8221 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     8281 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct
     8341 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     8401 ccgcaagccc ggtgccggat ccatgcccac gctactgcgg gtttatatag acggtcctca
     8461 cgggatgggg aaaaccacca ccacgcaact gctggtggcc ctgggttcgc gcgacgatat
     8521 cgtctacgta cccgagccga tgacttact
//
