LOCUS       sequence-490323-        6559 bp    DNA     circular SYN 23-OCT-2025
DEFINITION  Plasmid for PiggyBac /ITR mediated insertion of F-Tractin 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 6559)
  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 6559)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 23, 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-F-Tractin-mKate2
FEATURES             Location/Qualifiers
     source          1..6559
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   29..91
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(230..247)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(340..928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(420..439)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1099..1890,1891..1959))
                     /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     1722..1741
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1960..2064)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2365..2387
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2379..2396
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2380..2396
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2407..2426
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2407..2425
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   2476..2510
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        2818..3197
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3199..3475
                     /label=chicken beta-actin promoter
     intron          3476..4492
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4416..4438
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4500..4519
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    4555..4579
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4726..5421
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHR"
     protein_bind    complement(5437..5461)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(5506..5523)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5575..5595)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5993..6004)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6133..6150)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6139..6363
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 aaacctcgat atacagaccg ataaaacaca tgcgtcaatt ttacgcatga ttatctttaa
       61 cgtacgtcac aatatgatta tctttctagg gttaaacgcg tatgccgctt aattaactgg
      121 cctcatgggc cttccgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
      181 attaacatgg tcatagctgt ttccttgcgt attgggcgct ctccgcttcc tcgctcactg
      241 actcgctgcg ctcggtcgtt cgggtaaagc ctggggtgcc taatgagcaa aaggccagca
      301 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
      361 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
      421 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
      481 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
      541 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
      601 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
      661 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
      721 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
      781 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
      841 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
      901 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
      961 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     1021 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     1081 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     1141 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     1201 gggcttacca tctggcccca gtgctgcaat gataccgcga gaaccacgct caccggctcc
     1261 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     1321 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     1381 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     1441 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     1501 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     1561 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     1621 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     1681 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     1741 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     1801 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     1861 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     1921 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     1981 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     2041 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc
     2101 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     2161 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     2221 ggccgctaca gggcgctccc attcgccatt caggctgcgc aactgttggg aagggcgttt
     2281 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     2341 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc
     2401 gcgacgtaat acgactcact atagggcgaa ttggcggaag gccgtcaagg cctaggcgcg
     2461 ccgccactag attaacccta gaaagatagt ctgcgtaaaa ttgacgcatg cattcttgaa
     2521 atattgctct ctctttctaa atagcgcgaa tccgtcgctg tgcatttagg acatctcagt
     2581 cgccgcttgg agctcccgtg aggcgtgctt gtcaatgcgg taagtgtcac tgattttgaa
     2641 ctataacaac cgcgtgagtc aaaatgacgc atgattatct tttacgtgac ttttaagatt
     2701 taactcatac gataattata ttgttatttc gtgttctact tacgtgataa cttattatat
     2761 atatattttc ttgttataga tatcaactag aatcccgggc tagcatgggc ccatctcgac
     2821 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     2881 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     2941 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     3001 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     3061 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     3121 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     3181 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc
     3241 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
     3301 gggcgggggg gggggggggc gcgcgccggg cggggcgggg cggggcgagg ggcggggcgg
     3361 ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt
     3421 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt
     3481 cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg
     3541 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg
     3601 ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct
     3661 tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg
     3721 tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg
     3781 gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg
     3841 tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt
     3901 gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc
     3961 tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc
     4021 ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc
     4081 gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg
     4141 tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg
     4201 acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc
     4261 tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct
     4321 tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg
     4381 ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg
     4441 cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg
     4501 caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattcagat catcacaagt
     4561 ttgtacaaaa aagcaggctc cgcggccgcc cccttcacca tggcgaggcc tagaggcgct
     4621 ggcccttgtt ctcctggcct ggaaagagcc cctagaagaa gcgtgggcga gctgagactg
     4681 ctgttcgagg ctggcggctc tggatcggat ccaccggtcg ctaccgtgag cgagctgatt
     4741 aaggagaaca tgcacatgaa gctgtacatg gagggcaccg tgaacaacca ccacttcaag
     4801 tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggcg
     4861 gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctaccagctt catgtacggc
     4921 agcaaaacct tcatcaacca cacccagggc atccccgact tctttaagca gtccttcccc
     4981 gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc
     5041 caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac
     5101 ttcccatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc ctccaccgag
     5161 accctgtacc ccgctgacgg cggcctggaa ggcagagccg acatggccct gaagctcgtg
     5221 ggcgggggcc acctgatctg caacttgaag accacataca gatccaagaa acccgctaag
     5281 aacctcaaga tgcccggcgt ctactatgtg gacagaagac tggaaagaat caaggaggcc
     5341 gacaaagaga cctacgtcga gcagcacgag gtggctgtgg ccagatactg cgacctccct
     5401 agcaaactgg ggcacagatg aagggtgggc gcgccgaccc agctttcttg tacaaagtgg
     5461 tgatgatctg cggccgcctc gagatgcatc gataatctag gatccctata gtgtcaccta
     5521 aattcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     5581 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     5641 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     5701 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     5761 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     5821 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     5881 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     5941 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     6001 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     6061 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc aggatccgag
     6121 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     6181 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     6241 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     6301 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     6361 tggcttctga ggcggaaaga acctgcagcc caagcttagt ttaaacacta gttaaaagtt
     6421 ttgttacttt atagaagaaa ttttgagttt ttgttttttt ttaataaata aataaacata
     6481 aataaattgt ttgttgaatt tattattagt atgtaagtgt aaatataata aaacttaata
     6541 tctattcaaa ttaataaat
//
