LOCUS       sequence-450445-        4982 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac cargo vector encoding protective nicking sgRNA marked with 
            blasticidin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4982)
  AUTHORS   Loveless BT, Carlson CK, Dentzel Helmy CA, Hu VJ, Ross SK, Demelo 
            MC, Murtaza A, Liang G, Ficht M, Singhai A, Pajoh-Casco MJ, Liu CC
  TITLE     Open-ended molecular recording of sequential cellular events into 
            DNA
  JOURNAL   bioRxiv 2021.11.05.467507
REFERENCE   2  (bases 1 to 4982)
  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 
            2021.11.05.467507"
COMMENT     Sequence Label: pTBL3346 PB-nicking guide-blast
FEATURES             Location/Qualifiers
     source          1..4982
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        622..870
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     622..642
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     793..812
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    1407..1524
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1593..1804
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1824..1833
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1830..2228
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(2240..2257)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2246..2453
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2660..2722
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(2773..2791)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(2831..3646)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2831..3646)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3554..3573
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3638..3657
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3871..4459
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4360..4379
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4571..4590
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4571..4589
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   4619..4653
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 ctgaactggc cgataattgc agacgatcga attcgagttg gcgcgcctgt cattctaaat
       61 ctctctttca gcctaaagct ttttccccgt atccccccag gtgtctgcag gctcaaagag
      121 cagcgagaag cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc
      181 ccgcacgctg ccggctcggg gatgcggggg gagcgccgga ccggagcgga gccccgggcg
      241 gctcgctgct gccccctagc gggggaggga cgtaattaca tccctggggg ctttgggagg
      301 gggctgtccc cgtgagctcc caggcgcgcc tgtcattcta aatctctctt tcagcctaaa
      361 gctttttccc cgtatccccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag
      421 aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg ctgccggctc
      481 ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct
      541 agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt ccccgtgagc
      601 tcaataaaag agcccacaac ggagggccta tttcccatga ttccttcata tttgcatata
      661 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
      721 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      781 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      841 ttatatatct tgtggaaagg acgaaacacc gactccagtc tttctagaag agtttaagag
      901 ctaagctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag
      961 tggcaccgag tcggtgcttt ttttgctgga aatctgctcg tcagtggtgc tcacactgac
     1021 gaatcatgta cagatcatac cgatgactgc ctggcgactc acaactaagc aagacagccg
     1081 gaaccagcgc cggcgaacac cactgcatat atggcatatc acaacagtcc aagcagttac
     1141 agagatgtta cgaaccctga acaggttgac tattaattgt gtctgcttga tgtggacacc
     1201 aggtggcgct ggacatcaga tttggagagg cagttgtcta gggaaccggg ctctgtgcca
     1261 gcgcaggagg caggctggct ctcctattcc agggatgctc atccaggaag gaaaggttgc
     1321 atgctggaca cactaacctt acagcggtgg gctctatggg gtacctacgt atagatcttg
     1381 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     1441 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     1501 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg
     1561 cgccggctcg agtggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     1621 cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt
     1681 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
     1741 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
     1801 acaggtgtcg tgacgcggga tccgccacca tggccaagcc tttgtctcaa gaagaatcca
     1861 ccctcattga aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg
     1921 tcgccagcgc agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt
     1981 ttactggggg accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg
     2041 gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg
     2101 gacggtgccg acaggtgctt ctcgatctgc atcctgggat caaagccata gtgaaggaca
     2161 gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg
     2221 agggctaact cgagcgacgc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2281 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2341 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2401 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     2461 ggctctatga gtatcgatac tagttaaaag ttttgttact ttatagaaga aattttgagt
     2521 ttttgttttt ttttaataaa taaataaaca taaataaatt gtttgttgaa tttattatta
     2581 gtatgtaagt gtaaatataa taaaacttaa tatctattca aattaataaa taaacctcga
     2641 tatacagacc gataaaacac atgcgtcaat tttacgcatg attatcttta acgtacgtca
     2701 caatatgatt atctttctag ggttaatcta gtatacgcgt atgcggccgc ttaattaatc
     2761 cagcttttgt tccctttagt gagggttaat tccgagcggc cgcgtgttac aaccaattaa
     2821 ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     2881 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     2941 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     3001 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     3061 gagtgacgac tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt
     3121 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccattattca
     3181 ttcgtgattg cgcctgagcg aggcgaaata cgcgatcgct gttaaaagga caattacaaa
     3241 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     3301 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     3361 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     3421 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     3481 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     3541 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     3601 ttaatcgcgg cctggagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     3661 tactgtttat gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa
     3721 tgtaacatca gagattttga gacacaacgt ggctttgttg aataaatcga acttttgctg
     3781 agttgaagga tcagtcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3841 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3901 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3961 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4021 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4081 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4141 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4201 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4261 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4321 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4381 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4441 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4501 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtgc
     4561 ggccgccaat taatacgact cactataggg cgaattgggg cgcgccattc tagattaacc
     4621 ctagaaagat agtctgcgta aaattgacgc atgcattctt gaaatattgc tctctctttc
     4681 taaatagcgc gaatccgtcg ctgtgcattt aggacatctc agtcgccgct tggagctccc
     4741 gtgaggcgtg cttgtcaatg cggtaagtgt cactgatttt gaactataac gaccgcgtga
     4801 gtcaaaatga cgcatgatta tcttttacgt gacttttaag atttaactca tacgataatt
     4861 atattgttat ttcatgttct acttacgtga taacttatta tatatatatt ttcttgttat
     4921 agatatcaac tagaatgcta gcatgggccc agatctccat gggccctgaa ttcgcatcta
     4981 ga
//
