LOCUS       sequence-298844-        5969 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5969)
  AUTHORS   Chiang HR, Schoenfeld LW, Ruby JG, Auyeung VC, Spies N, Baek D, 
            Johnston WK, Russ C, Luo S, Babiarz JE, Blelloch R, Schroth GP, 
            Nusbaum C, Bartel DP
  TITLE     Mammalian microRNAs: experimental evaluation of novel and previously
            annotated genes.
  JOURNAL   Genes Dev. 2010 May 15. 24(10):992-1009.
  PUBMED    20413612
REFERENCE   2  (bases 1 to 5969)
  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: "Genes Dev. 
            2010 May 15. 24(10):992-1009."
COMMENT     Sequence Label: pcDNA3.2/V5 hsa-mir-150
FEATURES             Location/Qualifiers
     source          1..5969
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    677..701
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1175..1199)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1225..1266
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1306..1325)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1350..1398
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1600..2028
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1687..1706)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1897..1918
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2037..2057)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2042..2371
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2222..2357
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2284..2303
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2438..3232
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2492..2511)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3102..3121
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3408..3529
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3445..3464)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3499..3518
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3578..3594)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3578..3594)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3591..3613)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3602..3618
                     /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)."
     promoter        complement(join(3626..3632,3633..3650,3651..3656))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3671..3692
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3809..3826)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3980..4568)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4060..4079)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4739..5530,5531..5599))
                     /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     5362..5381
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5600..5704)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5779..5798)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagttaag ctatcaacaa gtttgtacaa aaaagcaggc tcatatggga gtgggtgtgc
      721 agtttctgcg actcagggtg gcgtcccccc aacctgtccc tgccccttcc tgccctcttt
      781 gatgcggccc cacttcctct ggcaggaacc cccgccctcc ctggacctgg gtataaggca
      841 gggactgggc ccacggggag gcagcgtccc cgaggcagca gcggcagcgg cggctcctct
      901 ccccatggcc ctgtctccca acccttgtac cagtgctggg ctcagaccct ggtacaggcc
      961 tgggggacag ggacctgggg accccggcac cggcaggccc caaggggtga ggtgagcggg
     1021 cattgggacc tcccctccct gtactcccat ctctgctgcg gcttttatgc gtctctcccc
     1081 ttcgggtccc acatatcctc tggtgcgctc ctgcctcacc gcccccaccc catgcctgtc
     1141 gtccccacct ctgtgtgatg cgcaaagtaa gcttacccag ctttcttgta caaagtggtt
     1201 gatctagagg gcccgcggtt cgaaggtaag cctatcccta accctctcct cggtctcgat
     1261 tctacgcgta ccggttagta atgagtttaa acgggggagg ctaactgaaa cacggaagga
     1321 gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg
     1381 gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata
     1441 ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc
     1501 ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca
     1561 tagcagatct gcgcagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     1621 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1681 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1741 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1801 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1861 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1921 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1981 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     2041 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2101 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     2161 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     2221 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     2281 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     2341 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     2401 cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     2461 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     2521 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     2581 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     2641 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     2701 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     2761 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     2821 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     2881 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     2941 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     3001 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     3061 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     3121 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     3181 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     3241 ctggggttcg cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     3301 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     3361 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     3421 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3481 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     3541 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3601 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3661 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3721 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3781 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3841 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3901 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3961 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4021 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4081 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4141 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4201 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4261 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4321 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4381 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4441 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4501 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4561 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4621 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4681 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4741 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4801 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4861 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4921 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4981 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5041 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5101 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5161 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5221 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5281 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5341 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5401 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5461 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5521 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5581 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5641 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5701 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     5761 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     5821 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     5881 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     5941 tcgcgatgta cgggccagat atacgcgtt
//
