LOCUS       sequence-439868-        7843 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology directed repair template for knocking in mGreenLantern 
            reporter to TFAP2C..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7843)
  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 7843)
  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_TFAP2C-T2A-mGreenLantern-Hygro
FEATURES             Location/Qualifiers
     source          1..7843
                     /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             2171..2224
                     /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(2225..2227,2228..2230,2231..2944)
                     /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(2270..2291)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2945..2976
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        3026..3525
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3061..3084)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3458..3477
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3545..4570
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVHRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(4592..4609)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4598..4822
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    4870..4901
                     /label=rox
                     /bound_moiety="Dre recombinase"
     CDS             6258..6830
                     /codon_start=1
                     /gene="Corynebacterium diphtheriae tox"
                     /product="diphtheria toxin fragment A"
                     /label=DTA
                     /translation="DDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYD
                     DDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKEL
                     GLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEI
                     NFETRGKRGQDAMYEYMAQACAGNRVRR"
     intron          6907..6972
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     rep_origin      complement(7255..7843)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7335..7354)
                     /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 tttccccgag tagttgagct gccctttcat tattgtgtta gcatatctta caaaaatgtg
     1201 ttgaaaaccc gatttgtttt gtcacaacca gtaaagagag attgtgtgcc ttctacctcc
     1261 tgatagcaag acttttgtct ttcaaatgta aagatcaggg tggatttgtg ggcaaattaa
     1321 ctgtctccaa aacgacttaa tgcagctgtg ggttttctca tagaaaccaa cagtaagttg
     1381 atttgaaaat gatggtaaat gaagcagaac tagtcgagcc aggaaatggg gtaaaagaga
     1441 gcgcagtgaa ccaggctgtg tcaggagtca gaatttcagg gggatgcttc cttctcccgg
     1501 ttcctcacgg aatttgttta ttggaaccaa cttgtaccaa ggtaaactgt cccttaagct
     1561 catgggttgg aatccaggcc ccattacttt taccagtcct ggtgaccttg gccagttagc
     1621 tggggttaag ttaatcctgt tgtcttttct aattgcagtg aggattaaat aagatagtgc
     1681 tttaaaacag cttaactcag agcctggcgc agaatagttg ctaagcgcag cagttggcta
     1741 ttagtattaa attctccttc ctcgggttga agcagttgtg cttgcctccc gggcgccagc
     1801 ctcagtgtag ttggttctgt gctcttgacc tgtaaggagc tagatggaac tcatcgagtc
     1861 aactgactgt cttcacaggc aactgtgtaa agaattcaca gaacttctca gccaagaccg
     1921 gacaccccat gggaccagca ggctcgcccc agtcttggag acgaacatac agaactgctt
     1981 gtctcatttc agcctgatta cccacgggtt tggcagccag gccatctgtg ccgcggtgtc
     2041 tgccctgcag aactacatca aagaagccct gattgtcata gacaaatcct acatgaaccc
     2101 tggagaccag agtccagctg attctaacaa aaccctggag aaaatggaga aacacaggaa
     2161 aggctctggc gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg
     2221 cccaatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     2281 ggacggcgac gtaaacggcc acaagttcag cgtccgcggc gagggcgagg gcgatgccac
     2341 caacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     2401 caccctcgtg accaccttag gctacggcgt ggcctgcttc gcccgctacc ccgaccacat
     2461 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     2521 ctctttcaag gacgacggta cctacaagac ccgcgccgag gtgaagttcg agggcgacac
     2581 cctggtgaac cgcatcgtgc tgaagggcat cgacttcaag gaggacggca acatcctggg
     2641 gcacaagctg gagtacaact tcaacagcca caaggtctat atcacggccg acaagcagaa
     2701 gaacggcatc aaggctaact tcaagacccg ccacaacgtt gaggacggcg gcgtgcagct
     2761 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     2821 ccactacctg agccatcagt ccaaactgag caaagacccc aacgagaagc gcgatcacat
     2881 ggtcctgaag gagagggtga ccgccgccgg gattacacat gacatggacg agctgtacaa
     2941 gtaataactt taaataatgc caattattta aagttacata tgagaaagta taggaacttc
     3001 agcttgatat cgaattaatt ctaccgggta ggggaggcgc ttttcccaag gcagtctgga
     3061 gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc
     3121 acacattcca catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg
     3181 ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc
     3241 aggacgtgac aaatggaagt agcacgtctc actagtctcg tgcagatgga cagcaccgct
     3301 gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct
     3361 ttctgggctc agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg
     3421 ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc
     3481 acgtctgccg cgctgttctc ctcttcctca tctccgggcc tttcgacctg cagccaacgc
     3541 caccatgaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt
     3601 cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt
     3661 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa
     3721 agatcgttat gttcatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga
     3781 cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac
     3841 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat
     3901 ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca
     3961 aggaatcggt caatacacta cgtggcgtga tttcatatgc gcgattgctg atccccatgt
     4021 gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga
     4081 tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt
     4141 cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga
     4201 ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt
     4261 ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc
     4321 gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt
     4381 tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc
     4441 cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga
     4501 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc
     4561 aaaggaatag gctctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     4621 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     4681 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     4741 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     4801 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctcgacatac
     4861 tagagtactt aactttaaat aatgccaatt atttaaagtt aggcgcgccg atatctagaa
     4921 aggttaggag agtagggaag gaacaggact gcaaaaatcc ttctccaccg cacagactgg
     4981 gaacccctcc tggcctgggg gaagagtttg ttacctacct tactatttaa agagccttca
     5041 ctggttctgc atcacccgcc cctggacttc ttagttgttt ctctagcgct gagctatctc
     5101 ctaactttgg acctattatc agaaggtgac aagtactggc tctttattca ttaagctttt
     5161 tttttttgaa ccccattctt tccttctctg aaagtggtgc tataagtttt agaatctttt
     5221 aaatacattc cctgggccaa cagacccaca cacttagcca ttgaaatgtc aaattgatgt
     5281 gccctagatc aacagatcaa caataccttt ttttcagtgt taaggtaatg gttggttttt
     5341 gtgtccgcta aatatttacc ttgaaaaaaa gaaaagtgtg tatctagctt cttcagagat
     5401 caagtcctct ggtaggaggc aaaggttcta tctgcttagc aactagttaa taagtggtat
     5461 ctgacacact ctaaaccccg tgttcaaacg ggggccttct ggttttagga aacttgtaga
     5521 aacgaagcct gctgattgat ttttttctcc tttttttttt tttttttttt ttaactttga
     5581 aagttaactc ttcaaatggg agactctttg aaatgacatg ttcctttaag gtactgaagc
     5641 tttatttgca tatttatttc agatgtttcg agtaaacttg aaaagggtag gcacgaagca
     5701 atttgttgct gcttgtcacc cccaagtccc cgtggaggtt ctgtatttta agaaacagtg
     5761 cgttgagtgt acagatttta tttatgcgta atttaatggg gtctgtaaat actggtgcac
     5821 ttcttacgac ttttttgaga catgggatcc aattttaata ttaactttta atggtgatgg
     5881 ggtaatctat aacacatcat aaggttttat tcatatatat acagggtatt aagaattaag
     5941 aggatgctgg gctctacctc cagatcttgt cagtgtggtt ttcaagagga agcaaaaagc
     6001 ctctccaccc aggcctggaa tgtttccacc caatgtcgag cagtgtggtt ttgcaagagg
     6061 aagcaaaaag cctctccacc caggcctgga atgtttccac ccaatgtcga gcaaaccccg
     6121 cccagcgtct tgtcattggc gaattcgaac acgcagatgc agtcggggcg gcgcggtccc
     6181 aggtccactt cgcatattaa ggtgacgcgt gtggcctcga acaccgagcg accctgcagg
     6241 tcctcgccat ggatcctgat gatgttgttg attcttctaa atcttttgtg atggaaaact
     6301 tttcttcgta ccacgggact aaacctggtt atgtagattc cattcaaaaa ggtatacaaa
     6361 agccaaaatc tggtacacaa ggaaattatg acgatgattg gaaagggttt tatagtaccg
     6421 acaataaata cgacgctgcg ggatactctg tagataatga aaacccgctc tctggaaaag
     6481 ctggaggcgt ggtcaaagtg acgtatccag gactgacgaa ggttctcgca ctaaaagtgg
     6541 ataatgccga aactattaag aaagagttag gtttaagtct cactgaaccg ttgatggagc
     6601 aagtcggaac ggaagagttt atcaaaaggt tcggtgatgg tgcttcgcgt gtagtgctca
     6661 gccttccctt cgctgagggg agttctagcg ttgaatatat taataactgg gaacaggcga
     6721 aagcgttaag cgtagaactt gagattaatt ttgaaacccg tggaaaacgt ggccaagatg
     6781 cgatgtatga gtatatggct caagcctgtg caggaaatcg tgtcaggcga tctctttgtg
     6841 aaggaacctt acttctgtgg tgtgacataa ttggacaaac tacctacaga gatttaaagc
     6901 tctaaggtaa atataaaatt tttaagtgta taatgtgtta aactactgat tctaattgtt
     6961 tgtgtatttt agattccaac ctatggaact gatgaatggg agcagtggtg gaatgcagat
     7021 ccactagagt cgagggatgc agaaattgat gatctattaa acaataaaga tgtccactaa
     7081 aatggaagtt tttcctgtca tactttgtta agaagggtga gaacagagta cctacatttt
     7141 gaatggaagg attggagcta cgggggtggg ggtggggtgg gattagataa atgcctgctc
     7201 tttactgaag gctctttact attgctttat gataatgttt catagttgga tgtttttcca
     7261 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     7321 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     7381 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     7441 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     7501 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     7561 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     7621 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     7681 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     7741 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     7801 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caa
//
