LOCUS       sequence-66026-e        6201 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 6201)
  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 6201)
  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 dme-let-7
FEATURES             Location/Qualifiers
     source          1..6201
                     /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(1314..1338)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1691..1732
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1772..1791)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1816..1864
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2066..2494
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2153..2172)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2363..2384
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2503..2523)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2508..2837
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2688..2823
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2750..2769
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2904..3698
                     /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(2958..2977)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3568..3587
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3874..3995
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3911..3930)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3965..3984
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4044..4060)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4044..4060)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4057..4079)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4068..4084
                     /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(4092..4098,4099..4116,4117..4122))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4137..4158
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4275..4292)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4446..5034)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4526..4545)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5205..5996,5997..6065))
                     /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     5828..5847
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6066..6170)
                     /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 aggcttcaaa agcattttta atatgatttc
      961 tccgtattat ttttcttctt gtttgccatc atcgtttcaa ccaaaaaccg aaccaatgat
     1021 atccagaaga tcctttaaat accaaaccac ctagcaaaaa ggactacacc aaggaccttt
     1081 ttctctctgg caaattgagg tagtaggttg tatagtagta attacacatc atactataca
     1141 atgtgctagc tttctttgct tgactacaag ccgcatttga taaaagaatc ccaatcgaac
     1201 tgcaccactt aataaaccca atccccagcc atacaaaagt tggtggtgca acaaattttg
     1261 attgtggaca acaaagaaag tgttctgagc caaactattg ttaaatatca ttaacccagc
     1321 tttcttgtac aaagtggttg atctagaggg cccgcagtgg ggtcagcttc taccggggcg
     1381 gcgtcccggg gtcttggaac tgcatgcaga ctgcctgctt gggaaacata cttctttata
     1441 tgcccatatg gacctgctaa gctatggaat gtaaagaagt atgtatctca ggccgggacc
     1501 tctctcgccg cactgagggg cactccacac cacgggggcc gccgcggccg acagccagcc
     1561 aggcggctgt ggtgggggtg aggctggggc ggcaggctgc ctgggcgttg gtggacggtc
     1621 gtgtccccgc cagcattgtt ctgtcacttt gcctgtgtca gcaggtggaa gtgctcacca
     1681 gacattcgaa ggtaagccta tccctaaccc tctcctcggt ctcgattcta cgcgtaccgg
     1741 ttagtaatga gtttaaacgg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     1801 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacgggtgt tgggtcgttt
     1861 gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc
     1921 cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt
     1981 gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc agatctgcgc
     2041 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2101 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2161 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2221 ggcatccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2281 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2341 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2401 atctcggtct attcttttga tttataaggg attttgggga tttcggccta ttggttaaaa
     2461 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     2521 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2581 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2641 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2701 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     2761 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     2821 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     2881 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     2941 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     3001 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3061 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     3121 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3181 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3241 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3301 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3361 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3421 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     3481 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     3541 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     3601 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     3661 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgcgaa
     3721 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     3781 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     3841 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     3901 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     3961 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     4021 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4081 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4141 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4201 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4261 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4321 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4381 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4441 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4501 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4561 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4621 ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4681 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4741 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4801 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4861 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     4921 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4981 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5041 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5101 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5161 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5221 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5281 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5341 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5401 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5461 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5521 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5581 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5641 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5701 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5761 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5821 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5881 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5941 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6001 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6061 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6121 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6181 cgaaaagtgc cacctgacgt c
//
