LOCUS       sequence-66022-e        6221 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 6221)
  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 6221)
  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 cel-mir-60
FEATURES             Location/Qualifiers
     source          1..6221
                     /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(1334..1358)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1711..1752
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1792..1811)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1836..1884
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2086..2514
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2173..2192)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2383..2404
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2523..2543)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2528..2857
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2708..2843
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2770..2789
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2924..3718
                     /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(2978..2997)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3588..3607
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3894..4015
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3931..3950)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3985..4004
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4064..4080)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4064..4080)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4077..4099)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4088..4104
                     /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(4112..4118,4119..4136,4137..4142))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4157..4178
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4295..4312)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4466..5054)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4546..4565)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5225..6016,6017..6085))
                     /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     5848..5867
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6086..6190)
                     /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 aggcttggtg ataggcaacc atcttttctg
      961 tgtctcaact atctaccatt ccataagccc acctactttt gtgtcactat aaaagagaat
     1021 tgtggtgtag gtgataacag tgctctttgt attattatct acaattactt cgtattcata
     1081 ttctactcga aaaccgcttg ttcttgaact ggaagagtgc cataaaatca tgacaaagta
     1141 cgtgatatta tgcacatttt ctagttcaag acttgagaaa tcggttaact gtttttgaaa
     1201 caacttctga tttttgttga cggtagtttt tgctatcatg agttccgggt aaatatcata
     1261 tactgtaaga catttgttag aaatcacaat gtttctttga ttgattattt caatttgctt
     1321 atgggaaact cttacccagc tttcttgtac aaagtggttg atctagaggg cccgcagtgg
     1381 ggtcagcttc taccggggcg gcgtcccggg gtcttggaac tgcatgcaga ctgcctgctt
     1441 gggaaacata cttctttata tgcccatatg gacctgctaa gctatggaat gtaaagaagt
     1501 atgtatctca ggccgggacc tctctcgccg cactgagggg cactccacac cacgggggcc
     1561 gccgcggccg acagccagcc aggcggctgt ggtgggggtg aggctggggc ggcaggctgc
     1621 ctgggcgttg gtggacggtc gtgtccccgc cagcattgtt ctgtcacttt gcctgtgtca
     1681 gcaggtggaa gtgctcacca gacattcgaa ggtaagccta tccctaaccc tctcctcggt
     1741 ctcgattcta cgcgtaccgg ttagtaatga gtttaaacgg gggaggctaa ctgaaacacg
     1801 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     1861 gcacgggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     1921 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     1981 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     2041 ctgccatagc agatctgcgc agctggggct ctagggggta tccccacgcg ccctgtagcg
     2101 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2161 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2221 cccgtcaagc tctaaatcgg ggcatccctt tagggttccg atttagtgct ttacggcacc
     2281 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     2341 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     2401 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgggga
     2461 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     2521 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2581 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     2641 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     2701 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     2761 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     2821 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     2881 cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     2941 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     3001 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     3061 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     3121 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     3181 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     3241 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     3301 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     3361 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     3421 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     3481 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     3541 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     3601 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     3661 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     3721 gggactctgg ggttcgcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     3781 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     3841 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     3901 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     3961 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4021 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4081 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4141 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4201 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4261 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4321 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4381 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4441 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4501 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4561 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4621 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
     4681 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4741 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4801 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4861 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     4921 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4981 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5041 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5101 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5161 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5221 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5281 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5341 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5401 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5461 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5521 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5581 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5641 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5701 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5761 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5821 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5881 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5941 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6001 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6061 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6121 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6181 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
