LOCUS       sequence-416794-        8327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes one forebrain neuron specific transcription factor under the
            control of a TetOn promoter at the CLYBL safe harbor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8327)
  AUTHORS   Agarwal D, Dash N, Mazo KW, Chopra M, Avila MP, Patel A, Wong RM, 
            Jia C, Do H, Cheng J, Chiang C, Jurlina SL, Roshan M, Perry MW, Rho 
            JM, Broyer R, Lee CD, Weinreb RN, Gavrilovici C, Oesch NW, Welsbie 
            DS, Wahlin KJ
  TITLE     Human retinal ganglion cell neurons generated by synchronous BMP 
            inhibition and transcription factor mediated reprogramming.
  JOURNAL   NPJ Regen Med. 2023 Sep 29;8(1):55. doi: 10.1038/s41536-023-00327-x.
  PUBMED    37773257
REFERENCE   2  (bases 1 to 8327)
  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: "NPJ Regen 
            Med."; date: "2023-09-29"; volume: "8(1):55. doi"; pages: " 
            10.1038/s41536-023-00327-x"
COMMENT     Sequence Label: P932_CLYBL-CBX3-Cbh-Zeo-TetO-NEUROG2
FEATURES             Location/Qualifiers
     source          1..8327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..241
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(449..466)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(620..1208)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(700..719)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1379..2170,2171..2239))
                     /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     2002..2021
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2240..2344)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2419..2438)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    2608..2641
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     enhancer        4117..4402
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          4632..4859
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      4865..4874
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4871..5242
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     CDS             5252..5308
                     /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             5309..6016
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGWSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     CDS             6017..6064
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    6083..6203
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     6173..6192
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    6288..6306
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6324..6342
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6360..6378
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6396..6414
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6432..6450
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6468..6486
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     6531..6555
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     polyA_signal    7384..7608
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cggaccatac tatctagaaa tagtttaaga gctaagctgg aaacagcata gcaagtttaa
      301 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttggcggc
      361 cgcgacccca actggggtaa cctttgagtt ctctcagttg gggtccatag ggataacagg
      421 gtaatttgat atcgcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
      481 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
      541 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
      601 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
      661 gacgcacaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
      721 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
      781 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
      841 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
      901 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
      961 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     1021 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     1081 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     1141 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     1201 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     1261 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     1321 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     1381 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     1441 ttgcctgact cccggtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     1501 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     1561 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     1621 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     1681 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     1741 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     1801 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     1861 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     1921 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     1981 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     2041 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     2101 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     2161 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     2221 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     2281 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     2341 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     2401 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     2461 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     2521 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     2581 tcgtagggat aacagggtaa ttcatgggtg ccagggcgtg cccttgggct ccccgggcgc
     2641 gtaaagcttc gatgtacggg ccagatatag gaccatacta tctagaaata tggagaccag
     2701 caggagaatc atgccaatgg gccaatatac attctgaccc acagtttcat aataaaataa
     2761 aatggttgtg gttgtaagcc actatgtttc agagtggttt gttacacagc aataaataac
     2821 taatatagta ggcataccat caagtccaaa gtaggtagag aagaatgtaa atagcagagc
     2881 aaaacagcat gactggtggc tgggaggctt aaaactggga caggatcaga gtcatgaaag
     2941 aagtcaaaga aatggttcag aagtaaggct gagactgact tacaaaagct gaaagtccct
     3001 ttaagttggt gtttggtgca ttggcagggg caggtatggt gacttaaaag agccatgctc
     3061 aacaagatca agcacaacac aatcacgggt caccccagca gaccttagcg agtctagcca
     3121 tttctttggt ggtggtcaca gtcatgcttc agcccagttt ccacttggac aaatggtaca
     3181 tattttcaat gagatgaaaa ttaagataca atccatgtgc tcagagagtg atcacagctc
     3241 tgactaaaca ctgtgcccca aagtgttgag gaattgggaa aacctagctg agttagtggt
     3301 ctcttttctg ttacaataaa gctcataatg aaaattagcc ttctttgttc ttccccaagt
     3361 ctttctttct agacgaaact actttcaact gttggaactt ccttactgtt aacttccccc
     3421 gggaggtggt ccctgcagtt acgccaatga taacccccgc cagaaaaatc ttagtagcct
     3481 tccctttttg ttttccgtgc cccaactcgg cggattgact cggccccttc cggaaacacc
     3541 cgaatcaact tctagtcaaa ttattgttca cgccgcaatg acccacccct ggcccgcgtc
     3601 tgtggaactg acccctggtg tacaggagag ttcgctgctg aaagtggtcc caaaggggta
     3661 ctagttttta agctcccaac tccccctccc ccagcgtctg gaggattcca caccctcgca
     3721 ccgcaggggc gaggaagtgg gcggagtccg gttttggcgc cagccgctga ggctgccaag
     3781 cagaaaagcc accgctgagg agactccggt cactgtcctc gccccgcctc ccccttccct
     3841 ccccttgggg accaccgggc gccacgccgc gaacggtaag tgccgcggtc gtcggcgcct
     3901 ccgccctccc cctagggccc caattcccag cgggcgcggc gcgcggcccc tccccccgcc
     3961 gggcgcgcgc ccgctgcccc gcccttcgtg gccgcccggc gtgggcggtg ccacccctcc
     4021 ccccggcggc cccgcgcgca gctcccggct ccctccccct tcggatgtgg cttgagctgt
     4081 aggcgcggag ggccggacgc gtgctctaga ggtacccgtt acataactta cggtaaatgg
     4141 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaatagtaa cgccaatagg
     4201 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     4261 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     4321 ctggcattgt gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     4381 attattcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
     4441 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
     4501 gatgggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc
     4561 tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg
     4621 cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct
     4681 cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg
     4741 gcccttctcc tccgggctgt aattagctga gcaagaggta agggtttaag ggatggttgg
     4801 ttggtggggt attaatgttt aattacctgg agcacctgcc tgaaatcact ttttttcagg
     4861 ttgggccgcc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc
     4921 cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga
     4981 cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt
     5041 ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga
     5101 gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat
     5161 cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca
     5221 cttcgtggcc gaggagcagg acggaagtgg agctactaac ttcagcctac ttaagcaggc
     5281 tggagacgtg gaagagaacc ctggacctgt gtctaagggc gaagagctga ttaaggagaa
     5341 catgcacatg aagctgtaca tggagggcac cgtggacaac catcacttca agtgcacatc
     5401 cgagggcgaa ggcaagccct acgagggcac ccagaccatg agaatcaagg tggtcgaggg
     5461 cggccctctc cccttcgcct tcgacatcct ggctactagc ttcctctacg gcagcaagac
     5521 cttcatcaac cacacccagg gcatccccga cttcttcaag cagtccttcc ctgagggctt
     5581 cacatgggag agagtcacca catacgaaga cgggggcgtg ctgaccgcta cccaggacac
     5641 cagcctccag gacggctgcc tcatctacaa cgtcaagatc agaggggtga acttcacatc
     5701 caacggccct gtgatgcaga agaaaacact cggctgggag gccttcaccg agacgctgta
     5761 ccccgctgac ggcggcctgg aaggcagaaa cgacatggcc ctgaagctcg tgggctggag
     5821 ccatctgatc gcaaacgcca agaccacata tagatccaag aaacccgcta agaacctcaa
     5881 gatgcctggc gtctactatg tggactacag actggaaaga atcaaggagg ccaacaacga
     5941 gacctacgtc gagcagcacg aggtggcagt ggccagatac tgcgacctcc ctagcaaact
     6001 ggggcacaag cttaataaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa
     6061 aaaggaattc taactagagc ccaacttgtt tattgcagct tataatggtt acaaataaag
     6121 caaagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     6181 tccaaactca tcaatgtatc ttatcatgtc ggggattctc ctaaatcaag atagaatgtg
     6241 gcttgagctg taggcgcgga gggccggacg cgtgctctag agtttactcc ctatcagtga
     6301 tagagaacgt atgcagactt tactccctat cagtgataga gaacgtataa ggagtttact
     6361 ccctatcagt gatagagaac gtatgaccag tttactccct atcagtgata gagaacgtat
     6421 ctacagttta ctccctatca gtgatagaga acgtatatcc agtttactcc ctatcagtga
     6481 tagagaacgt ataagcttta ggcgtgtacg gtgggcgcct ataaaagcag agctcgttta
     6541 gtgaaccgtc agatcgccac catgttcgtc aaatccgaga ccttggagtt gaaggaggaa
     6601 gaggacgtgt tagtgctgct cggatcggcc tcccccgcct tggcggccct gaccccgctg
     6661 tcatccagcg ccgacgaaga agaggaggag gagccgggcg cgtcaggcgg ggcgcgtcgg
     6721 cagcgcgggg ctgaggccgg gcagggggcg cggggcggcg tggctgcggg tgcggagggc
     6781 tgccggcccg cacggctgct gggtctggta cacgattgca aacggcgccc ttcccgggcg
     6841 cgggccgtct cccgaggcgc caagacggcc gagacggtgc agcgcatcaa gaagacccgt
     6901 agactgaagg ccaacaaccg cgagcgaaac cgcatgcaca acctcaacgc ggcactggac
     6961 gcgctgcgcg aggtgctccc cacgttcccc gaggacgcca agctcaccaa gatcgagacc
     7021 ctgcgcttcg cccacaacta catctgggca ctcaccgaga ccctgcgcct ggcggatcac
     7081 tgcgggggcg gcggcggggg cctgccgggg gcgctcttct ccgaggcagt gttgctgagc
     7141 ccgggaggcg ccagcgccgc cctgagcagc agcggagaca gcccctcgcc cgcctccacg
     7201 tggagttgca ccaacagccc cgcgccgtcc tcctccgtgt cctccaattc cacctccccc
     7261 tacagctgca ctttatcgcc cgccagcccg gccgggtcag acatggacta ttggcagccc
     7321 ccacctcccg acaagcaccg ctatgcacct cacctcccca tagccaggga ttgtatctag
     7381 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     7441 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     7501 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     7561 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct agatagtatg
     7621 gtcagactgt tatttcttga cttttaaatg taagatatta tctactgact tccttctatg
     7681 taagatgagg attgagctct cttacccttc tcccatttcc tcatccttcc aacataaata
     7741 tattttggga ttatatcaac attcaatgtt acttaaagtg accttgtaaa tattttcaca
     7801 actgagccat gtttgatttg tatacttatg tttactttac tgtttttcct gaagttaata
     7861 attgccttga atttatttat ttctttaaaa atgtttcatt actcaggact gtagtttaca
     7921 ttacgattct ttgtgttata cagttgatgg gtttcttttc tttcttaatt tctttaaaaa
     7981 atagagatgg ggtcttacta tattacccag gctggtcttg aagtcctggg ctcaagtgat
     8041 cttcctgtct cagcctacca agtagctgag actataggtg caaaaaagcc actatacctg
     8101 gctagtttac aggttttaac aaatgcatta tgccacgtat ccattattac aggatcacac
     8161 aagatatttt cattaccctg aaagcatccc tgtgttccac catatttcta gatagtatgg
     8221 tctttggtac caagctttgc gcggatgtat gtgtgtgcgt gtacgtgtgt atgtgaagat
     8281 accgcctagg cgcctaggga attcgcggcc gccttttgct cacatgt
//
