LOCUS       sequence-439893-        8213 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology directed repair template for knocking in mGreenLantern 
            reporter to FIGLA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8213)
  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 8213)
  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: pHDR_FIGLA-T2A-mGreenLantern-PuroTK
FEATURES             Location/Qualifiers
     source          1..8213
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2224..2277
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(2278..2280,2281..2283,2284..2997)
                     /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(2323..2344)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2998..3029
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        3079..3578
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3114..3137)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3511..3530
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      3592..3601
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3598..4197
                     /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     4097..4117
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4204..5199
                     /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"
     primer_bind     complement(5221..5238)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5227..5451
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    5499..5530
                     /label=rox
                     /bound_moiety="Dre recombinase"
     CDS             6628..7200
                     /codon_start=1
                     /gene="Corynebacterium diphtheriae tox"
                     /product="diphtheria toxin fragment A"
                     /label=DTA
                     /translation="DDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYD
                     DDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKEL
                     GLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEI
                     NFETRGKRGQDAMYEYMAQACAGNRVRR"
     intron          7277..7342
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     rep_origin      complement(7625..8213)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7705..7724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgtc acttgtatag tggaaagact cgatcaagat tagaaaaaaa aagctactca
     1201 tacatatttt gtgtcagaga ctgctcctgg cagtgtatac atattaacct atttaatttt
     1261 cacaaccacc ctgtgctata caaacaatta tatccatttt acatgtgagg aaactaagga
     1321 acagaagtaa cttgcccaat acctgcaagt cagtgaagtg ttggggttgg gggggatgtg
     1381 ggtgatagtg ttaagtcctt tttaaccact ttcgttctaa caaaactcaa ctttgaaatg
     1441 taaaggaggg tggactagaa aatagataaa tgtttaaggc taagttaaag catttgattg
     1501 cggtgcttta ccatccacag tgatgtacat aataggtttc agtatccaaa atgcctgacc
     1561 atgacttgag cggctttatt tttcagtcat gggtttattt tttcttcaag ttgacagttt
     1621 cagaaaattg agcagagaca aaatgatctt gccacgttag taaaaccact gatggaaata
     1681 cactaaaatc cttcccattt cttttccttt gcaactaaaa taaaaaatta tcatcaggta
     1741 cataacattg tgtgcctgtt cttgattttt ctatcactgg catgtttatg tgacccatta
     1801 ggtgggacaa tttttttact gttggccact atgttctcag ttttttgttc tcttaaacgt
     1861 gacaaataac cgatgtgttt agccctttct gtattttgta ttgtttttgc aggatagatt
     1921 cccagaagta gaactgctga gtcacaggta ggtcattcta taacctgatg catagtgcca
     1981 atttataaca ccactggctg tataataaga gtgctaattc catcacattc ttagcaatga
     2041 gctctattaa aagaaggaat cttaatgggt aaaaaagtag tatctttaaa tttaaacatc
     2101 tttgattact aataaaatta attttttgca tatttcttta tttcttttgt aaacggttct
     2161 tatttttaac tatttataat ggaatgtttt ctctgtttca gacttccaca agtaggctct
     2221 ggcgagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg
     2281 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     2341 gacgtaaacg gccacaagtt cagcgtccgc ggcgagggcg agggcgatgc caccaacggc
     2401 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     2461 gtgaccacct taggctacgg cgtggcctgc ttcgcccgct accccgacca catgaagcag
     2521 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catctctttc
     2581 aaggacgacg gtacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2641 aaccgcatcg tgctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2701 ctggagtaca acttcaacag ccacaaggtc tatatcacgg ccgacaagca gaagaacggc
     2761 atcaaggcta acttcaagac ccgccacaac gttgaggacg gcggcgtgca gctcgccgac
     2821 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2881 ctgagccatc agtccaaact gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2941 aaggagaggg tgaccgccgc cgggattaca catgacatgg acgagctgta caagtaataa
     3001 ctttaaataa tgccaattat ttaaagttac atatgagaaa gtataggaac ttcagcttga
     3061 tatcgaatta attctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg
     3121 ctttagcagc cccgctgggc acttggcgct acacaagtgg cctctgccct cgcacacatt
     3181 ccacatccac cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt
     3241 ctactcctcc cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt
     3301 gacaaatgga agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat
     3361 ggaagcgggt aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg
     3421 ctcagaggct gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg
     3481 cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg
     3541 ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac ctgcagccaa cgccaccatg
     3601 gggaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta
     3661 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgacccggac
     3721 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     3781 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     3841 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     3901 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     3961 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     4021 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     4081 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     4141 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgccgga
     4201 tccatgccca cgctactgcg ggtttatata gacggtcctc acgggatggg gaaaaccacc
     4261 accacgcaac tgctggtggc cctgggttcg cgcgacgata tcgtctacgt acccgagccg
     4321 atgacttact ggcaggtgct gggggcttcc gagacaatcg cgaacatcta caccacacaa
     4381 caccgcctcg accagggtga gatatcggcc ggggacgcgg cggtggtaat gacaagcgcc
     4441 cagataacaa tgggcatgcc ttatgccgtg accgacgccg ttctggctcc tcatatcggg
     4501 ggggaggctg ggagctcaca tgccccgccc ccggccctca ccctcatctt cgaccgccat
     4561 cccatcgccg ccctcctgtg ctacccggcc gcgcgatacc ttatgggcag catgaccccc
     4621 caggccgtgc tggcgttcgt ggccctcatc ccgccgacct tgcccggcac aaacatcgtg
     4681 ttgggggccc ttccggagga cagacacatc gaccgcctgg ccaaacgcca gcgccccggc
     4741 gagcggcttg acctggctat gctggccgcg attcgccgcg tttacgggct gcttgccaat
     4801 acggtgcggt atctgcaggg cggcgggtcg tggcgggagg attggggaca gctttcgggg
     4861 acggccgtgc cgccccaggg tgccgagccc cagagcaacg cgggcccacg accccatatc
     4921 ggggacacgt tatttaccct gtttcgggcc cccgagttgc tggcccccaa cggcgacctg
     4981 tacaacgtgt ttgcctgggc cttggacgtc ttggccaaac gcctccgtcc catgcacgtc
     5041 tttatcctgg attacgacca atcgcccgcc ggctgccggg acgccctgct gcaacttacc
     5101 tccgggatgg tccagaccca cgtcaccacc cccggctcca taccgacgat ctgcgacctg
     5161 gcgcgcacgt ttgcccggga gatgggggag gctaactgag ctctagagct cgctgatcag
     5221 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     5281 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     5341 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     5401 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     5461 cggaaagaac cagctggggc tcgacatact agagtactta actttaaata atgccaatta
     5521 tttaaagtta ggcgcgccga tatctagcag ggttaccttc tagagacaaa taaatgcagt
     5581 cttgaaagtt ttggagtctc tctttttttt tttttttttg agatagagtt tcactcttat
     5641 cacccaggct ggagtggcac ggtgcgatct cggctcactg caacctccgc ctcctgggtt
     5701 caagtgattc tcctgcctca gcctcctgag tagctgggat tacaggcatg cgccaccacg
     5761 cccagctaat ttttgtattt ttagtagaga cagggcttct ccacgttggt caggctggtc
     5821 tcgaactccc gacctcaggt gattcaacca cctcagcctc ccaaagtgct gggattacag
     5881 gaatgagcca ctgtgccccg tgggagtctc ttttttaaaa aagaagtttt gtagtaattc
     5941 cgagtttaac aattaccctt ttaaatagcc ttgcctctct caagtttact ccactctgcc
     6001 ttctccttaa cttcatgctt tgaagaaata ggcttcaggg gcttcatcct ttgcttccaa
     6061 ctcattgtca tcctctgggg acctacatct ttttttcatt gctttgagaa ataagtgact
     6121 gttacccttc caatagaaat taaccttaat agaaatgaag acttaagcta aaagagttgt
     6181 aaacattggc caaggggagt gagagttttc aagggtttct agatacccag ctaaggccaa
     6241 atggcacctt ttgtgcaagg tagacagcca aacaccttga gcacaaagac tttgtcttta
     6301 taaccattgc tcacaaagac atagtacctc cagatcttgt cagtgtggtt ttcaagagga
     6361 agcaaaaagc ctctccaccc aggcctggaa tgtttccacc caatgtcgag cagtgtggtt
     6421 ttgcaagagg aagcaaaaag cctctccacc caggcctgga atgtttccac ccaatgtcga
     6481 gcaaaccccg cccagcgtct tgtcattggc gaattcgaac acgcagatgc agtcggggcg
     6541 gcgcggtccc aggtccactt cgcatattaa ggtgacgcgt gtggcctcga acaccgagcg
     6601 accctgcagg tcctcgccat ggatcctgat gatgttgttg attcttctaa atcttttgtg
     6661 atggaaaact tttcttcgta ccacgggact aaacctggtt atgtagattc cattcaaaaa
     6721 ggtatacaaa agccaaaatc tggtacacaa ggaaattatg acgatgattg gaaagggttt
     6781 tatagtaccg acaataaata cgacgctgcg ggatactctg tagataatga aaacccgctc
     6841 tctggaaaag ctggaggcgt ggtcaaagtg acgtatccag gactgacgaa ggttctcgca
     6901 ctaaaagtgg ataatgccga aactattaag aaagagttag gtttaagtct cactgaaccg
     6961 ttgatggagc aagtcggaac ggaagagttt atcaaaaggt tcggtgatgg tgcttcgcgt
     7021 gtagtgctca gccttccctt cgctgagggg agttctagcg ttgaatatat taataactgg
     7081 gaacaggcga aagcgttaag cgtagaactt gagattaatt ttgaaacccg tggaaaacgt
     7141 ggccaagatg cgatgtatga gtatatggct caagcctgtg caggaaatcg tgtcaggcga
     7201 tctctttgtg aaggaacctt acttctgtgg tgtgacataa ttggacaaac tacctacaga
     7261 gatttaaagc tctaaggtaa atataaaatt tttaagtgta taatgtgtta aactactgat
     7321 tctaattgtt tgtgtatttt agattccaac ctatggaact gatgaatggg agcagtggtg
     7381 gaatgcagat ccactagagt cgagggatgc agaaattgat gatctattaa acaataaaga
     7441 tgtccactaa aatggaagtt tttcctgtca tactttgtta agaagggtga gaacagagta
     7501 cctacatttt gaatggaagg attggagcta cgggggtggg ggtggggtgg gattagataa
     7561 atgcctgctc tttactgaag gctctttact attgctttat gataatgttt catagttgga
     7621 tgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     7681 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     7741 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     7801 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     7861 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     7921 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     7981 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     8041 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     8101 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     8161 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caa
//
