LOCUS       sequence-158396-        4496 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty entry vector for cloning inducible mammalian expression and an
            in-frame N-terminal 3xFLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4496)
  AUTHORS   Bajikar SS, Wang CC, Borten MA, Pereira EJ, Atkins KA, Janes KA
  TITLE     Tumor-Suppressor Inactivation of GDF11 Occurs by Precursor 
            Sequestration in Triple-Negative Breast Cancer.
  JOURNAL   Dev Cell. 2017 Nov 20;43(4):418-435.e13. doi: 
            10.1016/j.devcel.2017.10.027.
  PUBMED    29161592
REFERENCE   2  (bases 1 to 4496)
  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"; doi: 
            "10.1016/j.devcel.2017.10"; journalName: "Dev Cell"; date: 
            "2017-11-20- 20"; volume: "43"; issue: "4"; pages: "418-435.e13"
COMMENT     Sequence Label: pEN_TT miRc2 3xFLAG
FEATURES             Location/Qualifiers
     source          1..4496
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..48
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    67..166
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        198..512
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    206..224
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    242..260
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    277..295
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    313..331
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    349..367
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    384..402
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    420..438
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     485..509
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             521..586
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     ncRNA           618..739
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     CDS             775..1080
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRIVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     959..978
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     ncRNA           1101..1227
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     protein_bind    complement(1266..1365)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1477..1496)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1555..1574)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(2148..2681)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(2870..2875,2876..2892,2893..2898))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      3555..4143
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4044..4063
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      4308..4394
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      join(4486..4496,1..17)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctgacggatg gcctttttgc gtttctacaa actcttcctg ttagttagtt acttaagctc
       61 gggccccaaa taatgatttt attttgactg atagtgacct gttcgttgca acaaattgat
      121 aagcaatgct tttttataat gccaactttg tacaaaaaag caggctttaa aggaaccaat
      181 tcagtcgact ggatcccgag tttactccct atcagtgata gagaacgtat gtcgagttta
      241 ctccctatca gtgatagaga acgatgtcga gtttactccc tatcagtgat agagaacgta
      301 tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg
      361 atagagaacg tatgtcgagt ttatccctat cagtgataga gaacgtatgt cgagtttact
      421 ccctatcagt gatagagaac gtatgtcgag gtaggcgtgt acggtgggag gcctatataa
      481 gcagagctcg tttagtgaac cgtcagatcg ccactagatg gactacaaag accatgacgg
      541 tgattataaa gatcatgaca tcgattacaa ggatgacgat gacaagacta gtcgactagg
      601 gataacaggg taattgtttg aatgaggctt cagtacttta cagaatcgtt gcctgcacat
      661 cttggaaaca cttgctggga ttacttcttc aggttaaccc aacagaaggc tcgagaaggt
      721 atattgctgt tgacagtgag cgtgctgcag gtaagcttga ggtgtgcttc cagaatgcag
      781 tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga tgtacagagt
      841 gatattattg acacgcccgg gcgacggata gtgatccccc tggccagtgc acgtctgctg
      901 tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc
      961 atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga agtggctgat
     1021 ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatatag
     1081 aattccaacc tgctgcttgc ctactgcctc ggaattcaag gggctacttt aggagcaatt
     1141 atcttgttta ctaaaactga ataccttgct atctctttga tacattttta caaagctgaa
     1201 ttaaaatggt ataaattaaa tcactttttt caattggaag actaatgcgt tttcgagata
     1261 tctagaccca gctttcttgt acaaagttgg cattataaga aagcattgct tatcaatttg
     1321 ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt atttgccatc cagctgcagc
     1381 tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat
     1441 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
     1501 aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     1561 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc
     1621 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     1681 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     1741 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat
     1801 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     1861 ccctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     1921 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     1981 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     2041 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     2101 acgaggggaa attaggccgg gaagccgatc tcggcttgaa cgaattgtta ggtggcggta
     2161 cttgggtcga tatcaaagtg catcacttct tcccgtatgc ccaactttgt atagagagcc
     2221 actgcgggat cgtcaccgta atctgcttgc acgtagatca cataagcacc aagcgcgttg
     2281 gcctcatgct tgaggagatt gatgagcgcg gtggcaatgc cctgcctccg gtgctcgccg
     2341 gagactgcga gatcatagat atagatctca ctacgcggct gctcaaacct gggcagaacg
     2401 taagccgcga gagcgccaac aaccgcttct tggtcgaagg cagcaagcgc gatgaatgtc
     2461 ttactacgga gcaagttccc gaggtaatcg gagtccggct gatgttggga gtaggtggct
     2521 acgtctccga actcacgacc gaaaagatca agagcagccc gcatggattt gacttggtca
     2581 gggccgagcc tacatgtgcg aatgatgccc atacttgagc cacctaactt tgttttaggg
     2641 cgactgccct gctgcgtaac atcgttgctg ctgcgtaaca tcgttgctgc tccataacat
     2701 caaacatcga cccacggcgt aacgcgcttg ctgcttggat gcccgaggca tagactgtac
     2761 aaaaaaacag tcataacaag ccatgaaaac cgccactgcg ccgttaccac cgctgcgttc
     2821 ggtcaaggtt ctggaccagt tgcgtgagcg catacgctac ttgcattaca gtttacgaac
     2881 cgaacaggct tatgtcaact gggttcgtgc cttcatccgt ttccacggtg tgcgtcaccc
     2941 ggcaaccttg ggcagcagcg aagtcgaggc atttctgtcc tggctggcga acgagcgcaa
     3001 ggtttcggtc tccacgcatc gtcaggcatt ggcggccttg ctgttcttct acggcaaggt
     3061 gctgtgcacg gatctgccct ggcttcagga gatcggaaga cctcggccgt cgcggcgctt
     3121 gccggtggtg ctgaccccgg atgaagtggt tcgcatcctc ggttttctgg aaggcgagca
     3181 tcgtttgttc gcccaggact ctagctatag ttctagtggt tggctactaa taggttgtat
     3241 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     3301 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     3361 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt
     3421 ggttaattgg ttgtaacact ggcagagcat tacgctgact tgacgggacg gcgcaagctc
     3481 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3541 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3601 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3661 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     3721 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3781 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3841 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3901 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3961 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4021 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4081 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4141 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4201 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc tagcatggat
     4261 ctcggggacg tctaactact aagcgagagt agggaactgc caggcatcaa ataaaacgaa
     4321 aggctcagtc ggaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
     4381 tgagtaggac aaatccgccg ggagcggatt tgaacgttgt gaagcaacgg cccggagggt
     4441 ggcgggcagg acgcccgcca taaactgcca ggcatcaaac taagcagaag gccatc
//
