LOCUS       sequence-494476-        6500 bp    DNA     circular SYN 03-DEC-2025
DEFINITION  Plasmid for PiggyBac /ITR mediated insertion of pm-mKate2 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 6500)
  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 6500)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 3, 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-pm-mKate2
FEATURES             Location/Qualifiers
     source          1..6500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..28)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(121..709)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(201..220)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(880..1671,1672..1740))
                     /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     1503..1522
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1741..1845)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2146..2168
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2160..2177
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2161..2177
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2188..2207
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2188..2206
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   2257..2291
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        2599..2978
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2980..3257
                     /label=chicken beta-actin promoter
     intron          3258..4274
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4198..4220
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4282..4301
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    4337..4361
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4442..5140
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     protein_bind    complement(5159..5183)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(5228..5245)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5297..5317)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5715..5726)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5855..5872)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5861..6085
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6310..6372
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 tctccgcttc ctcgctcact gactcgctgc gctcggtcgt tcgggtaaag cctggggtgc
       61 ctaatgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
      121 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
      181 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      241 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      301 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      361 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      421 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      481 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      541 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      601 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      661 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      721 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      781 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      841 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
      901 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
      961 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     1021 agaaccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     1081 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1141 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1201 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1261 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1321 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1381 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1441 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     1501 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1561 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1621 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1681 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     1741 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     1801 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     1861 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     1921 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     1981 tagaccgaga tagggttgag tggccgctac agggcgctcc cattcgccat tcaggctgcg
     2041 caactgttgg gaagggcgtt tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     2101 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
     2161 gtaaaacgac ggccagtgag cgcgacgtaa tacgactcac tatagggcga attggcggaa
     2221 ggccgtcaag gcctaggcgc gccgccacta gattaaccct agaaagatag tctgcgtaaa
     2281 attgacgcat gcattcttga aatattgctc tctctttcta aatagcgcga atccgtcgct
     2341 gtgcatttag gacatctcag tcgccgcttg gagctcccgt gaggcgtgct tgtcaatgcg
     2401 gtaagtgtca ctgattttga actataacaa ccgcgtgagt caaaatgacg catgattatc
     2461 ttttacgtga cttttaagat ttaactcata cgataattat attgttattt cgtgttctac
     2521 ttacgtgata acttattata tatatatttt cttgttatag atatcaacta gaatcccggg
     2581 ctagcatggg cccatctcga cattgattat tgactagtta ttaatagtaa tcaattacgg
     2641 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     2701 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     2761 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     2821 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     2881 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     2941 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct
     3001 gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt
     3061 aattattttg tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg
     3121 ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg
     3181 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
     3241 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
     3301 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
     3361 gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt
     3421 ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg
     3481 gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc
     3541 ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg
     3601 aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag
     3661 gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc
     3721 tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg
     3781 ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg
     3841 gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg
     3901 gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt
     3961 aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg
     4021 ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga
     4081 aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc
     4141 agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt
     4201 tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct
     4261 tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca
     4321 aagaattcag atcatcacaa gtttgtacaa aaaagcaggc tccgcggccg cccccttcac
     4381 catgctgtgc tgtatgagaa gaaccaaaca ggttgaaaag aatgatgagg accaaaagat
     4441 catggtgagc gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt
     4501 gaacaaccac cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca
     4561 gaccatgaga atcaaggcgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc
     4621 taccagcttc atgtacggca gcaaaacctt catcaaccac acccagggca tccccgactt
     4681 ctttaagcag tccttccccg agggcttcac atgggagaga gtcaccacat acgaagacgg
     4741 gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt
     4801 caagatcaga ggggtgaact tcccatccaa cggccctgtg atgcagaaga aaacactcgg
     4861 ctgggaggcc tccaccgaga ccctgtaccc cgctgacggc ggcctggaag gcagagccga
     4921 catggccctg aagctcgtgg gcgggggcca cctgatctgc aacttgaaga ccacatacag
     4981 atccaagaaa cccgctaaga acctcaagat gcccggcgtc tactatgtgg acagaagact
     5041 ggaaagaatc aaggaggccg acaaagagac ctacgtcgag cagcacgagg tggctgtggc
     5101 cagatactgc gacctcccta gcaaactggg gcacagatga agaagggtgg gcgcgccgac
     5161 ccagctttct tgtacaaagt ggtgatgatc tgcggccgcc tcgagatgca tcgataatct
     5221 aggatcccta tagtgtcacc taaattcaac ctctggatta caaaatttgt gaaagattga
     5281 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     5341 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     5401 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     5461 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     5521 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     5581 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     5641 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     5701 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     5761 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     5821 ccgcctcccc gcaggatccg agctcgctga tcagcctcga ctgtgccttc tagttgccag
     5881 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     5941 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     6001 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     6061 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaacctgcag cccaagctta
     6121 gtttaaacac tagttaaaag ttttgttact ttatagaaga aattttgagt ttttgttttt
     6181 ttttaataaa taaataaaca taaataaatt gtttgttgaa tttattatta gtatgtaagt
     6241 gtaaatataa taaaacttaa tatctattca aattaataaa taaacctcga tatacagacc
     6301 gataaaacac atgcgtcaat tttacgcatg attatcttta acgtacgtca caatatgatt
     6361 atctttctag ggttaaacgc gtatgccgct taattaactg gcctcatggg ccttccgctc
     6421 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaacatg gtcatagctg
     6481 tttccttgcg tattgggcgc
//
