LOCUS       sequence-313587-        5602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggybacTransposon-based expression control of Tet-on 3G, Puro and 
            Cas13d U6 gRNA backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5602)
  AUTHORS   Chang Y, Syahirah R, Oprescu SN, Wang X, Jung J, Cooper SH, 
            Torregrosa-Allen S, Elzey BD, Hsu AY, Randolph LN, Sun Y, Kuang S, 
            Broxmeyer HE, Deng Q, Lian X, Bao X
  TITLE     Chemically-defined generation of human hemogenic endothelium and 
            definitive hematopoietic progenitor cells.
  JOURNAL   Biomaterials. 2022 May 6;285:121569. doi: 
            10.1016/j.biomaterials.2022.121569.
  PUBMED    35567999
REFERENCE   2  (bases 1 to 5602)
  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: 
            "Biomaterials."; date: "2022-05-6"; volume: "285:121569. doi"; 
            pages: " 10.1016/j.biomaterials.2022.121569"
COMMENT     Sequence Label: PiggyBac EF1a-Tet-on 3G-Puro U6 BbsI
FEATURES             Location/Qualifiers
     source          1..5602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     103..120
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    237..258
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(273..278,279..296,297..303)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    311..327
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     316..338
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     335..351
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     335..351
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   377..439
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     misc_RNA        complement(640..669)
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     promoter        complement(671..919)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(729..748)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(899..919)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     polyA_signal    920..1001
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(1022..1621)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1105..1125)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1602..1621
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(1631..1684)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(1694..2437)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             complement(2438..2461)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2461..2470
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2490..2701)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     polyA_signal    2882..3016
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   3292..3326
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3337..3354)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3337..3353)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3346..3368)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3562..3581)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3681..3703
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3741..3759)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3827..3931
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3932..4000,4001..4792)
                     /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(4150..4169)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4963..5551
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5452..5471
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
       61 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
      121 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
      181 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
      241 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
      301 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
      361 gccaaggtcg acttaaccct agaaagataa tcatattgtg acgtacgtta aagataatca
      421 tgcgtaaaat tgacgcatgt gttttatcgg tctgtatatc gaggtttatt tattaatttg
      481 aatagatatt aagttttatt atatttacac ttacatacta ataataaatt caacaaacaa
      541 tttatttatg tttatttatt tattaaaaaa aaacaaaaac tcaaaatttc ttctataaag
      601 taacaaaact tttaaaaaaa aaggtcttct cgaagacccg tttcaaaccc cgaccagttg
      661 gtaggggttc ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact
      721 ttcaagttac ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact
      781 acccaagaaa ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag
      841 tcaaattaat tccaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa
      901 tcatgggaaa taggccctcg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
      961 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taactaaacg ggccctctag
     1021 atcaggcacc gggcttgcgg gtcatgcacc aggtgcgcgg tccttcgggc acctcgacgt
     1081 cggcggtgac ggtgaagccg agccgctcgt agaaggggag gttgcggggc gcggaggtct
     1141 ccaggaaggc gggcaccccg gcgcgctcgg ccgcctccac tccggggagc acgacggcgc
     1201 tgcccagacc cttgccctgg tggtcgggcg agacgccgac ggtggccagg aaccacgcgg
     1261 gctccttggg ccggtgcggc gccaggaggc cttccatctg ttgctgcgcg gccagccggg
     1321 aaccgctcaa ctcggccatg cgcgggccga tctcggcgaa caccgccccc gcttcgacgc
     1381 tctccggcgt ggtccagacc gccaccgcgg cgccgtcgtc cgcgacccac accttgccga
     1441 tgtcgagccc gacgcgcgtg aggaagagtt cttgcagctc ggtgacccgc tcgatgtggc
     1501 ggtccggatc gacggtgtgg cgcgtggcgg ggtagtcggc gaacgcggcg gcgagggtgc
     1561 gtacggccct ggggacgtcg tcgcgggtgg cgaggcgcac cgtgggcttg tactcggtca
     1621 tctcgagcct agggccggga ttctcctcca cgtcaccgca tgttagaaga gaacctctgc
     1681 cctctccgct gcccccgggg agcatgtcaa ggtcaaaatc gtcaagagcg tcagcaggca
     1741 gcatatcaag gtcaaagtcg tcaagggcat cggctgggag catgtctaag tcaaaatcgt
     1801 caagggcgtc ggtcggcccg ccgctttcgc actttagctg tttctccagg ccacatatga
     1861 ttagttccag gccgaaaagg aaggcaggtt cggctccctg ccggtcgaac agctcaattg
     1921 cttgtttcag aagtgggggc atagaatcgg tggtaggtgt ctctctttcc tcttttgcta
     1981 cttgatgctc ctgttcctcc aatacgcagc ccagtgtaaa gtggcccacg gcggacagag
     2041 cgtacagtgc gttctccagg gagaagcctt gctgacacag gaacgcgagc tgattttcca
     2101 gggtttcgta ctgtttctct gttgggcggg tgccgagatg cactttagcc ccgtcgcgat
     2161 gtgagaggag agcacagcgg tatgacttgg cgttgttccg cagaaagtct tgccatgact
     2221 cgccttccag ggggcaggag tgggtatgat gcctgtccag catctcgatt ggcagggcat
     2281 cgagcagggc ccgcttgttc ttcacgtgcc agtacagggt aggctgctca actcccagct
     2341 tttgagcgag tttccttgtc gtcaggcctt cgataccgac tccattgagt aattccagag
     2401 cagagtttat gactttgctc ttgtccagtc tagacatctt atcgtcatcg tctttgtaat
     2461 ccatggtggc ggatcccgcg tcacgacacc tgtgttctgg cggcaaaccc gttgcgaaaa
     2521 agaacgttca cggcgactac tgcacttata tacggttctc ccccaccctc gggaaaaagg
     2581 cggagccagt acacgacatc actttcccag tttaccccgc gccaccttct ctaggcaccg
     2641 gttcaattgc cgacccctcc ccccaacttc tcggggactg tgggcgatgt gcgctctgcc
     2701 cactagtaga ccacctcccc tgcgagctaa gctggacagc caatgacggg taagagagtg
     2761 acatttttca ctaacctaag acaggagggc cgtcagagct actgcctaat ccaaagacgg
     2821 gtaaaagtga taaaaatgta tcactccaac ctaagacagg cgcagcttcc gagggatttg
     2881 agatccagac atgataagat acattgatga gtttggacaa accaaaacta gaatgcagtg
     2941 aaaaaaatgc cttatttgtg aaatttgtga tgctattgcc ttatttgtaa ccattataag
     3001 ctgcaataaa caagtttgat atctataaca agaaaatata tatataataa gttatcacgt
     3061 aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa
     3121 aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt
     3181 accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc
     3241 acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca
     3301 attttacgca gactatcttt ctagggttaa gaattcactg gccgtcgttt tacaacgtcg
     3361 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     3421 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     3481 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3541 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     3601 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3661 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3721 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     3781 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3841 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3901 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3961 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4021 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4081 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4141 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4201 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4261 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4321 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4381 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4441 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     4501 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4561 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4621 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     4681 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4741 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     4801 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     4861 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4921 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4981 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5041 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5101 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5161 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5221 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5281 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5341 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5401 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5461 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5521 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5581 cctggccttt tgctggcctt tt
//
