LOCUS       sequence-485265-        6634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Sleeping Beauty vector for inducible expression of a chicken H4 
            targeting shRNA using a shortened mir30 sequence. Allows 
            co-selection with Puromycin..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6634)
  TITLE     A novel approach towards a histone replacement system in Tetrapods
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6634)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pHREP-KD-2-shH4
FEATURES             Location/Qualifiers
     source          1..6634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        295..607
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    303..321
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    339..357
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    374..392
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    410..428
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    446..464
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    481..499
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    517..535
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     580..604
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             678..1277
                     /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(678..697)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1174..1194
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     ncRNA           1457..1471
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     polyA_signal    1571..1692
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1582..1601)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1636..1655
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             join(2414..3031,3032..3037,3038..3073,3074..3076,3077..3112,
                     3113..3115,3116..3151,3152..3157)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKIINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             3164..3220
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3215..4015
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="GPMIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQG
                     RPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEE
                     HQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQD
                     IALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3275..3294)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3885..3904
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4070..4087)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4076..4300
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4631..4648)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4802..5390)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4882..4901)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5561..6352,6353..6421))
                     /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     6184..6203
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6422..6526)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggatccctat acagttgaag tcggaagttt acatacactt aagttggagt cattaaaact
       61 cgtttttcaa ctactccaca aatttcttgt taacaaacaa tagttttggc aagtcagtta
      121 ggacatctac tttgtgcatg acacaagtca tttttccaac aattgtttac agacagatta
      181 tttcacttat aattcactgt atcacaattc cagtgggtca gaagtttaca tacactaagt
      241 tcgactcctc tgcagaatgc ggcgatgttt cggtaagggg tccgctatct agacgagttt
      301 actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg
      361 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgagtttact ccctatcagt
      421 gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta
      481 tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta
      541 tgtcgattag gcgtgtacgg tgggcgccta taaaagcaga gctcgtttag tgaaccgtca
      601 gatcgcctgg agccaattcc aactcttttg ccttatacct actttccgta ccacttccta
      661 ccctcgaaag gcctctgatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
      721 acgtccccag ggctgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
      781 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
      841 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
      901 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
      961 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     1021 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtttcg cccgaccacc
     1081 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     1141 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     1201 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     1261 gcaagcccgg tgcctgattc gaaggcctgt cgtgaagctt ggggatcaat tctctagagc
     1321 tcgctgatca gcctcgagct agcgctaccg gactcagatc tcgagctcaa gcttcgaatt
     1381 ctgcagtcga cggtaccgat ccaagaaggt atattgctgt tgacagtgag cgccacggcc
     1441 atggacgtgg tctatagtga agccacagat gtatagacca cgtccatggc cgtgatgcct
     1501 actgcctcgg acttcaaggg ctacgatcgt acgggatcca gtcaggccaa gcttccatcg
     1561 atagacatga taagatacat tgatgagttt ggacaaacca caacaagaat gcagtgaaaa
     1621 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     1681 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg
     1741 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtacctaggc gagaccctgt
     1801 ctcacaaaat aaagtaagcc cggactgagt gcggaaaggc gggcctggcg ggtctggtct
     1861 ccccatgcgg gccaccagag gccctgcagc cttcagtcgc ttgaaggggt aatggcgctt
     1921 ccactcacaa acatggcgga cagagcgtgt gaacgagatg aacagcccct caaaaatatg
     1981 gccgccgagg ctggacggcc gtgccccagc agcaccgcct ccgcgcccca cgtgatctct
     2041 cgccgggcac agcgctgacc gcggaggtcc aaccggaaga atgtccggat tggacattcg
     2101 gaagagggcc cgccttccct ggggaatctc tgcgcacgcg cagaacgctt cgaccaatga
     2161 aaacacagga agccgtccgc gcaaccgcgt tgcgtcactt ctgccgcccc tgtttcaagg
     2221 tatatagccg tagacggaac ttcgcctttc tctcggcctt agcgccattt ttttgggtga
     2281 gtgttttttg gttcctgcgt tgggattccg tgtacaatcc atagacatct gacctcggca
     2341 cttagcatca tcacagcaaa ctaactgtag cctttctctc tttccctgta gaaacctctg
     2401 cacctgaggc cacatgtcca gactggacaa gagcaaaatc ataaacagcg ctctggaatt
     2461 actcaatgga gtcggtatcg aaggcctgac gacaaggaaa ctcgctcaaa agctgggagt
     2521 tgagcagcct accctgtact ggcacgtgaa gaacaagcgg gccctgctcg atgccctgcc
     2581 aatcgagatg ctggacaggc atcataccca cagctgcccc ctggaaggcg agtcatggca
     2641 agactttctg cggaacaacg ccaagtcata ccgctgtgct ctcctctcac atcgcgacgg
     2701 ggctaaagtg catctcggca cccgcccaac agagaaacag tacgaaaccc tggaaaatca
     2761 gctcgcgttc ctgtgtcagc aaggcttctc cctggagaac gcactgtacg ctctgtccgc
     2821 cgtgggccac tttacactgg gctgcgtatt ggaggaacag gagcatcaag tagcaaaaga
     2881 ggaaagagag acacctacca ccgattctat gcccccactt ctgaagcaag caattgagct
     2941 gttcgaccgg cagggagccg aacctgcctt ccttttcggc ctggaactaa tcatatgtgg
     3001 cctggagaaa cagctaaagt gcgaaagcgg cgggccgacc gacgcccttg acgattttga
     3061 cttagacatg ctcccagccg atgcccttga cgactttgac cttgatatgc tgcctgctga
     3121 cgctcttgac gattttgacc ttgacatgct ccccgggtcc ggcgctacta acttcagcct
     3181 gctgaagcag gctggtgacg tcgaggagaa tcctggtccc atgattgaac aagatggatt
     3241 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     3301 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     3361 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     3421 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3481 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     3541 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3601 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3661 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     3721 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     3781 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     3841 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     3901 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     3961 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgattcga
     4021 aggcctgtcg tgaagcttgg ggatcaattc tctagagctc gctgatcagc ctcgactgtg
     4081 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     4141 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     4201 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     4261 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     4321 agctgggcct actagctact cgggacccct taccgaaaca tcgccgcatt ctgcagagga
     4381 gtcgaactta gtgtatgtaa acttctgacc cactgggaat gtgatgaaag aaataaaagc
     4441 tgaaatgaat cattctctct actattattc tgatatttca cattcttaaa ataaagtggt
     4501 gatcctaact gacctaagac agggaatttt tactaggatt aaatgtcagg aattgtgaaa
     4561 aagtgagttt aaatgtattt ggctaaggtg tatgtaaact tccgacttca actgtatagg
     4621 gatccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4681 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4741 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4801 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4861 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4921 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4981 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5041 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5101 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5161 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5221 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5281 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5341 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5401 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5461 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5521 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5581 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5641 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5701 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5761 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5821 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5881 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5941 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6001 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6061 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6121 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6181 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6241 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6301 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6361 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6421 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6481 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6541 aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     6601 aggaaattgt aagcgttaat attttgttaa aatt
//
