LOCUS       sequence-267400-        5799 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 5799)
  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 5799)
  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 mmu-mir-122
FEATURES             Location/Qualifiers
     source          1..5799
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     249..268
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        249..267
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    297..321
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(625..649)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             675..716
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(756..775)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    800..848
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1050..1478
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1137..1156)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1347..1368
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1487..1507)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1492..1821
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1672..1807
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1734..1753
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1888..2682
                     /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(1942..1961)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2552..2571
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2858..2979
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2895..2914)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2949..2968
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3028..3044)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3028..3044)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3041..3063)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3052..3068
                     /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(3076..3082,3083..3100,3101..3106))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3121..3142
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3259..3276)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3430..4018)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3510..3529)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4189..4980,4981..5049))
                     /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     4812..4831
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5050..5154)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5229..5248)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5420..5799
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
      241 atcgaaatta atacgactca ctatagggag acccaagctg gctagttaag ctatcaacaa
      301 gtttgtacaa aaaagcaggc tccctatgct ccagtttcag cgtttggaac cactgaggag
      361 tctgtgacag acaacggagg aatgtgaagg gaaggttaat acccttgtgc ctgacagact
      421 ttccttagca gagctgtgga gtgtgacaat ggtgtttgtg tccaaaccat caaacgccat
      481 tatcacacta aatagctact gctaggcaat ccgtccactc cacgcgtgac ttgacgtctg
      541 ttctctccga gcaagaagtt ctgtcttgtg ctagagttct ccattgtgtg taatgtatga
      601 ataaaatctg gtcctcttgt gcttacccag ctttcttgta caaagtggtt gatctagagg
      661 gcccgcggtt cgaaggtaag cctatcccta accctctcct cggtctcgat tctacgcgta
      721 ccggttagta atgagtttaa acgggggagg ctaactgaaa cacggaagga gacaataccg
      781 gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg gtgttgggtc
      841 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
      901 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
      961 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcagatct
     1021 gcgcagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     1081 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1141 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     1201 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     1261 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     1321 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     1381 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     1441 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     1501 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     1561 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     1621 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     1681 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     1741 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     1801 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     1861 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     1921 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     1981 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     2041 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     2101 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     2161 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     2221 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     2281 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     2341 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     2401 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     2461 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     2521 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     2581 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     2641 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     2701 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     2761 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     2821 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     2881 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     2941 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     3001 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     3061 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3121 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3181 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3241 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3301 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3361 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3421 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3481 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3541 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3601 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     3661 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3721 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3781 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3841 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     3901 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3961 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4021 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4081 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     4141 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     4201 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4261 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4321 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4381 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4441 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4501 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4561 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     4621 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     4681 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     4741 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     4801 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     4861 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4921 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4981 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5041 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5101 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5161 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
     5221 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
     5281 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
     5341 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
     5401 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
     5461 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     5521 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     5581 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     5641 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     5701 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     5761 tggcagtaca tctacgtatt agtcatcgct attaccatg
//
