LOCUS       sequence-490235-        6831 bp    DNA     circular SYN 22-OCT-2025
DEFINITION  Plasmid for PiggyBac /ITR mediated insertion of H2B-mEos sequence 
            downstream of CAG promoter using Gateway recombination cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6831)
  AUTHORS   Dady A, Davidson L, Loyer N, Rappich S, Findlay G, Sanders T, 
            Januschke J, Storey KG
  TITLE     Engineering fluorescent reporters in human pluripotent stem cells 
            and strategies for live imaging human neurogenesis.
  JOURNAL   Development. 2025 Aug 1:dev.205082. doi: 10.1242/dev.205082.
  PUBMED    40748212
REFERENCE   2  (bases 1 to 6831)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 22, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Development. 2025 Aug 1:dev.205082. doi: 10.1242/dev.205082."
COMMENT     Sequence Label: APX1-H2B-mEos3.2
FEATURES             Location/Qualifiers
     source          1..6831
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    83..307
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     296..313
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             442..453
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     851..871
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     923..940
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    985..1009
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(1741..2118)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     protein_bind    complement(2138..2162)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2198..2217)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          2225..3241
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(2279..2301)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     enhancer        3521..3900
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   4208..4242
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4292..4311)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4293..4311)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4322..4339)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4322..4338)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4331..4353)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        4654..4758
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4759..4827,4828..5619)
                     /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     complement(4977..4996)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5790..6378
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6279..6298
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6471..6488
                     /label=L4440
                     /note="L4440 vector, forward primer"
     repeat_region   6627..6689
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 caaaatttct tctataaagt aacaaaactt ttaactagtg tttaaactaa gcttgggctg
       61 caggttcttt ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg
      121 tcttcccaat cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac
      181 ctactcagac aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca
      241 ccttccaggg tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa
      301 ggcacagtcg aggctgatca gcgagctcgg atcctgcggg gaggcggccc aaagggagat
      361 ccgactcgtc tgagggcgaa ggcgaagacg cggaagaggc cgcagagccg gcagcaggcc
      421 gcgggaagga aggtccgctg gattgagggc cgaagggacg tagcagaagg acgtcccgcg
      481 cagaatccag gtggcaacac aggcgagcag ccaaggaaag gacgatgatt tccccgacaa
      541 caccacggaa ttgtcagtgc ccaacagccg agcccctgtc cagcagcggg caaggcaggc
      601 ggcgatgagt tccgccgtgg caatagggag ggggaaagcg aaagtcccgg aaaggagctg
      661 acaggtggtg gcaatgcccc aaccagtggg ggttgcgtca gcaaacacag tgcacaccac
      721 gccacgttgc ctgacaacgg gccacaactc ctcataaaga gacagcaacc aggatttata
      781 caaggaggag aaaatgaaag ccatacggga agcaatagca tgatacaaag gcattaaagc
      841 agcgtatcca catagcgtaa aaggagcaac atagttaaga ataccagtca atctttcaca
      901 aattttgtaa tccagaggtt gaatttaggt gacactatag ggatcctaga ttatcgatgc
      961 atctcgaggc ggccgcagat catcaccact ttgtacaaga aagctgggtc ggcgcgccca
     1021 cccttgtcgg cgcgcccacc cttttcatct tctggcgtta tcgggcaggc cagagtgggc
     1081 cacggcgtgc tcgtacagct tcactttgtt atagtccttg tcgtggctca ggatctcgat
     1141 gcagtggtcc acaaagtggg cgccaggcag cttcacgccc ttttctttgg ccttgtaggt
     1201 ggtccggaag tcgcatctgt agtgggcgtt cccttccagc agcagggcca tttcaatgtc
     1261 gccggtcagc acgccatccc gcacgtacat cttctcggtg ctgggctccc acttcagggt
     1321 ctttttctgc atcacggggc cgttggcggg gaagttggtg ccgtagaacc gcactttgtt
     1381 gtagaaggtg tcgccttcca tggtgatgtc gttccgggcg ttgcagatgc cgccgtcctc
     1441 aaaggtcagg ctccgctccc agctgtagcc cttggggaag ctctgcttaa agtagtcctg
     1501 gatgttgtcg gggtacttgg cgaacacccg gttgccgtag tggaaggcgg tggtcagaat
     1561 gtcgaaggcg aagggcaggg gtccgccctc tttcacttcc aggtccatgc tctgcttgcc
     1621 ctcaaagggc ttgccggtgc cgtcgccatc gatcacgaag tggtggccgt tcacgttgcc
     1681 ttccatccgc agcttgatct tcatgtcggg cttgatggcg ctggtggcga ccggtggatc
     1741 cttagcgctg gtgtacttgg tgatggcctt agtaccctcg gacacggcgt gcttggccaa
     1801 ctccccaggc agcagcaggc gcacggccgt ctggatctcc ctggaggtga tggtcgagcg
     1861 cttgttgtaa tgcgccaggc gggaagcctc acctgcgatg cgctcgaaaa tgtcgttcac
     1921 aaacgaattc atgatgccca tggccttgga cgaaatgccg gtgtcagggt ggacctgctt
     1981 cagaaccttg tacacataga tggaatagct ctccttgcgg ctgcgcttgc gcttcttgcc
     2041 gcctttcttc tgcgccttag tcaccgcctt cttggagccc tttttcgggg cgggagcaga
     2101 cttcgctggc tctggcatgt gaagggggcg gccgcggagc ctgctttttt gtacaaactt
     2161 gtgatgatct gaattctttg ccaaaatgat gagacagcac aataaccagc acgttgccca
     2221 ggagctgtag gaaaaagaag aaggcatgaa catggttagc agaggctcta gagccgccgg
     2281 tcacacgcca gaagccgaac cccgccctgc cccgtccccc ccgaaggcag ccgtcccccc
     2341 gcggacagcc ccgaggctgg agagggagaa ggggacggcg gcgcggcgac gcacgaaggc
     2401 cctccccgcc catttccttc ctgccggcgc cgcaccgctt cgccccgcgc ccgctagagg
     2461 gggtgcggcg gcgcctccca gatttcggct ccgcacagat ttgggacaaa ggaagtccct
     2521 gcgccctctc gcacgattac cataaaaggc aatggctgcg gctcgccgcg cctcgacagc
     2581 cgccggcgct ccgggggccg ccgcgcccct cccccgagcc ctccccggcc cgaggcggcc
     2641 ccgccccgcc cggcaccccc acctgccgcc accccccgcc cggcacggcg agccccgcgc
     2701 cacgccccgt acggagcccc gcacccgaag ccgggccgtg ctcagcaact cggggagggg
     2761 ggtgcagggg gggttgcagc ccgaccgacg cgcccacacc ccctgctcac ccccccacgc
     2821 acacaccccg cacgcagcct ttgttcccct cgcagccccc cccgcaccgc ggggcaccgc
     2881 ccccggccgc gctcccctcg cgcacactgc ggagcgcaca aagccccgcg ccgcgcccgc
     2941 agcgctcaca gccgccgggc agcgcggagc cgcacgcggc gctccccacg cacacacaca
     3001 cgcacgcacc ccccgagccg ctccccccgc acaaagggcc ctcccggagc ccctcaaggc
     3061 tttcacgcag ccacagaaaa gaaacaagcc gtcattaaac caagcgctaa ttacagcccg
     3121 gaggagaagg gccgtcccgc ccgctcacct gtgggagtaa cgcggtcagt cagagccggg
     3181 gcgggcggcg cgaggcggcg gcggagcggg gcacggggcg aaggcagcgc gcagcgactc
     3241 ccgcccgccg cgcgcctcgc tttttatagg gccgccgccg ccgccgcctc gccataaaag
     3301 gaaactttcg gagcgcgccg ctctgattgg ctgccgccgc acctctccgc ctcgcccccc
     3361 cccccccccc cccccccccc ccccccccct cccccccccc cccccccccc ccccccccca
     3421 tcgctgcaca aaataattaa aaaataaata aatacaaaat tgggggtggg gagggggggg
     3481 agatggggag agtgaagcag aacgtggggc tcacctcgac catggtaata gcgatgacta
     3541 atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg gcataatgcc
     3601 aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt
     3661 gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag
     3721 tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt
     3781 tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg
     3841 ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat aatcaatgtc
     3901 gagatgggcc catgctagcc cgggattcta gttgatatct ataacaagaa aatatatata
     3961 taataagtta tcacgtaagt agaacacgaa ataacaatat aattatcgta tgagttaaat
     4021 cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtt gttatagttc
     4081 aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca agcggcgact
     4141 gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga gcaatatttc
     4201 aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaatcta gtggcggcgc
     4261 gcctaggcct tgacggcctt ccgccaattc gccctatagt gagtcgtatt acgtcgcgct
     4321 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     4381 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgaaa
     4441 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg agcgccctgt agcggccact
     4501 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     4561 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     4621 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     4681 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     4741 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     4801 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     4861 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     4921 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     4981 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5041 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5101 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5161 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5221 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5281 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     5341 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     5401 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     5461 gccggtgagc gtggttctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     5521 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     5581 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     5641 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     5701 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     5761 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     5821 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     5881 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     5941 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6001 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6061 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6121 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6181 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6241 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6301 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6361 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6421 tggccttttg ctcattaggc accccaggct ttacccgaac gaccgagcgc agcgagtcag
     6481 tgagcgagga agcggagagc gcccaatacg caaggaaaca gctatgacca tgttaatgca
     6541 gctggcacga caggtttccc gactggaaag cgggcagtga gcggaaggcc catgaggcca
     6601 gttaattaag cggcatacgc gtttaaccct agaaagataa tcatattgtg acgtacgtta
     6661 aagataatca tgcgtaaaat tgacgcatgt gttttatcgg tctgtatatc gaggtttatt
     6721 tattaatttg aatagatatt aagttttatt atatttacac ttacatacta ataataaatt
     6781 caacaaacaa tttatttatg tttatttatt tattaaaaaa aaacaaaaac t
//
