LOCUS       sequence-117472-        6004 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 6004)
  AUTHORS   Auyeung VC, Ulitsky I, McGeary SE, Bartel DP
  TITLE     Beyond Secondary Structure: Primary-Sequence Determinants License 
            Pri-miRNA Hairpins for Processing.
  JOURNAL   Cell. 2013 Feb 14;152(4):844-58. doi: 10.1016/j.cell.2013.01.031.
  PUBMED    23415231
REFERENCE   2  (bases 1 to 6004)
  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.cell.2013.01"; journalName: "Cell"; date: "2013-02-14- 
            14"; volume: "152"; issue: "4"; pages: "844-58"
COMMENT     Sequence Label: pcDNA3.2 mir-1-1 reporter mir50-2
FEATURES             Location/Qualifiers
     source          1..6004
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    911..935
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1117..1141)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1494..1535
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1575..1594)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1619..1667
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1869..2297
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1956..1975)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2166..2187
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2306..2326)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2311..2640
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2491..2626
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2553..2572
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2707..3501
                     /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(2761..2780)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3371..3390
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3677..3798
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3714..3733)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3768..3787
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3847..3863)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3847..3863)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3860..3882)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3871..3887
                     /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(3895..3901,3902..3919,3920..3925))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3940..3961
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4078..4095)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4249..4837)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4329..4348)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5008..5799,5800..5868))
                     /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     5631..5650
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5869..5973)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagctatca acaagtttgt acaaaaaagc aggctctatt tttactaatt ctgaattcca
      961 taattctata ttctgtattc ctccccgccg gccgctgata tgtctggtat tcttgggttt
     1021 gaacttccag cgttgaaccc gcatattaga cgtatcgacg gccggcgggg cataatggct
     1081 ggtaagtttg gagagaccgg gggagtccca gagatgcccc agctttcttg tacaaagtgg
     1141 ttgatctaga gggcccgcag tggggtcagc ttctaccggg gcggcgtccc ggggtcttgg
     1201 aactgcatgc agactgcctg cttgggaaac atacttcttt atatgcccat atggacctgc
     1261 taagctatgg aatgtaaaga agtatgtatc tcaggccggg acctctctcg ccgcactgag
     1321 gggcactcca caccacgggg gccgccgcgg ccgacagcca gccaggcggc tgtggtgggg
     1381 gtgaggctgg ggcggcaggc tgcctgggcg ttggtggacg gtcgtgtccc cgccagcatt
     1441 gttctgtcac tttgcctgtg tcagcaggtg gaagtgctca ccagacattc gaaggtaagc
     1501 ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac cggttagtaa tgagtttaaa
     1561 cgggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg
     1621 gcaataaaaa gacagaataa aacgcacggg tgttgggtcg tttgttcata aacgcggggt
     1681 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc
     1741 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc
     1801 agccaacgtc ggggcggcag gccctgccat agcagatctg cgcagctggg gctctagggg
     1861 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1921 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     1981 tctcgccacg ttcgccggct ttccccgtca agctctaaat cggggcatcc ctttagggtt
     2041 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2101 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2161 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2221 tgatttataa gggattttgg ggatttcggc ctattggtta aaaaatgagc tgatttaaca
     2281 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     2341 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     2401 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2461 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     2521 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     2581 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     2641 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     2701 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     2761 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     2821 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     2881 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     2941 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     3001 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     3061 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     3121 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     3181 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     3241 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     3301 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     3361 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     3421 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     3481 cttcttgacg agttcttctg agcgggactc tggggttcgc gaaatgaccg accaagcgac
     3541 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     3601 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     3661 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     3721 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     3781 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     3841 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     3901 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     3961 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4021 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4081 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4141 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4201 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4261 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4321 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4381 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4441 tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4501 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4561 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4621 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4681 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4741 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4801 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4861 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4921 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4981 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5041 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5101 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5161 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5221 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5281 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5341 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5401 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5461 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5521 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5581 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5641 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5701 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5761 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5821 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5881 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5941 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6001 cgtc
//
