LOCUS       sequence-12091-e        4990 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector withTRE promoter driving mouse G beta 2 miR30-based 
            shRNA with GFP coexpression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4990)
  AUTHORS   Shin KJ, Wall EA, Zavzavadjian JR, Santat LA, Liu J, Hwang JI, 
            Rebres R, Roach T, Seaman W, Simon MI, Fraser ID
  TITLE     A single lentiviral vector platform for microRNA-based conditional 
            RNA interference and coordinated transgene expression.
  JOURNAL   Proc Natl Acad Sci U S A. 2006 Sep 12. 103(37):13759-64.
  PUBMED    16945906
REFERENCE   2  (bases 1 to 4990)
  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: "Proc Natl 
            Acad Sci U S A. 2006 Sep 12. 103(37):13759-64."
COMMENT     Sequence Label: pEN_TGmiR_Gb2-K
FEATURES             Location/Qualifiers
     source          1..4990
                     /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"
     protein_bind    join(205..223,224..246,247..265,266..288,289..307,308..330,
                     331..349,350..372,373..391,392..414,415..433,434..456,
                     457..475)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        508..546
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     543..567
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(653..655,656..658,659..1372)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(653..655,656..658,659..1369)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(698..719)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(959..978)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1306..1327
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           1404..1525
                     /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"
     ncRNA           1553..1567
                     /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"
     ncRNA           1595..1721
                     /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(1760..1859)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1971..1990)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2049..2068)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(2642..3175)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3364..3369,3370..3386,3387..3392))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      4049..4637
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4538..4557
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      4802..4888
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      join(4980..4990,1..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 ggatccttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt
      241 taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg
      301 atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt
      361 cgagtttacc actccctatc agtgatagag aaaagtgaaa gtcgagttta ccactcccta
      421 tcagtgatag agaaaagtga aagtcgagtt taccactccc tatcagtgat agagaaaagt
      481 gaaagtcgag ctcggtaccc gggtcgaggt aggcgtgtac ggtgggaggc ctatataagc
      541 agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc
      601 catagaagac accgggaccg atccagcctc cgcggccccg aactagtcca ccatggtgag
      661 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
      721 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
      781 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
      841 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
      901 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
      961 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     1021 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     1081 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     1141 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     1201 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     1261 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     1321 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aaactagtcg
     1381 actagggata acagggtaat tgtttgaatg aggcttcagt actttacaga atcgttgcct
     1441 gcacatcttg gaaacacttg ctgggattac ttcttcaggt taacccaaca gaaggctcga
     1501 gaaggtatat tgctgttgac agtgagcgcg ctcatgtatt cccacgacaa tagtgaagcc
     1561 acagatgtat tgtcgtggga atacatgagc atgcctactg cctcggaatt caaggggcta
     1621 ctttaggagc aattatcttg tttactaaaa ctgaatacct tgctatctct ttgatacatt
     1681 tttacaaagc tgaattaaaa tggtataaat taaatcactt ttttcaattg gaagactaat
     1741 gcgttttcga gatatctaga cccagctttc ttgtacaaag ttggcattat aagaaagcat
     1801 tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc
     1861 catccagctg cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa
     1921 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     1981 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
     2041 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     2101 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     2161 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     2221 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     2281 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     2341 tgcgctggca gtgtccctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     2401 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     2461 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     2521 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     2581 taaccttatt tttgacgagg ggaaattagg ccgggaagcc gatctcggct tgaacgaatt
     2641 gttaggtggc ggtacttggg tcgatatcaa agtgcatcac ttcttcccgt atgcccaact
     2701 ttgtatagag agccactgcg ggatcgtcac cgtaatctgc ttgcacgtag atcacataag
     2761 caccaagcgc gttggcctca tgcttgagga gattgatgag cgcggtggca atgccctgcc
     2821 tccggtgctc gccggagact gcgagatcat agatatagat ctcactacgc ggctgctcaa
     2881 acctgggcag aacgtaagcc gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa
     2941 gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta atcggagtcc ggctgatgtt
     3001 gggagtaggt ggctacgtct ccgaactcac gaccgaaaag atcaagagca gcccgcatgg
     3061 atttgacttg gtcagggccg agcctacatg tgcgaatgat gcccatactt gagccaccta
     3121 actttgtttt agggcgactg ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg
     3181 ctgctccata acatcaaaca tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga
     3241 ggcatagact gtacaaaaaa acagtcataa caagccatga aaaccgccac tgcgccgtta
     3301 ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg agcgcatacg ctacttgcat
     3361 tacagtttac gaaccgaaca ggcttatgtc aactgggttc gtgccttcat ccgtttccac
     3421 ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg aggcatttct gtcctggctg
     3481 gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg cattggcggc cttgctgttc
     3541 ttctacggca aggtgctgtg cacggatctg ccctggcttc aggagatcgg aagacctcgg
     3601 ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag tggttcgcat cctcggtttt
     3661 ctggaaggcg agcatcgttt gttcgcccag gactctagct atagttctag tggttggcta
     3721 ctaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
     3781 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     3841 tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
     3901 ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct gacttgacgg
     3961 gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4021 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4081 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4141 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     4201 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4261 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4321 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4381 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4441 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4501 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4561 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4621 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4681 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4741 ccgctagcat ggatctcggg gacgtctaac tactaagcga gagtagggaa ctgccaggca
     4801 tcaaataaaa cgaaaggctc agtcggaaga ctgggccttt cgttttatct gttgtttgtc
     4861 ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgtgaagca
     4921 acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaactaagca
     4981 gaaggccatc
//
