LOCUS       sequence-66019-e        6224 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 6224)
  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 6224)
  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-46
FEATURES             Location/Qualifiers
     source          1..6224
                     /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(1337..1361)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1714..1755
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1795..1814)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1839..1887
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2089..2517
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2176..2195)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2386..2407
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2526..2546)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2531..2860
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2711..2846
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2773..2792
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2927..3721
                     /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(2981..3000)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3591..3610
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3897..4018
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3934..3953)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3988..4007
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4067..4083)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4067..4083)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4080..4102)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4091..4107
                     /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(4115..4121,4122..4139,4140..4145))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4160..4181
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4298..4315)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4469..5057)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4549..4568)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5228..6019,6020..6088))
                     /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     5851..5870
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6089..6193)
                     /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 aggctacttc tcctgcagac cgtgcgcggg
      961 agggggtctt cagccattca ttcaatgtct caccttccaa ctacttttct tttctcacct
     1021 attccctatt taattgtatt ttcctatgta ttttgaacga aaagttgaag gtaagggtcc
     1081 aaactgctga ggtgaagctg aagagagccg tctattgaca gttcaagacc acgagtcgtt
     1141 gtgtgctgtc atggagtcgc tctcttcaga tgatccggtc aatggtattt tgttttaata
     1201 taatattttg caaaaaaaaa taatttgaat gaattttatt tcgaggagtt tttaatcgaa
     1261 aagttcacat taaaatattg aaagttccga aactctaaat ttgaaaacaa ttcttaaaat
     1321 cttcgcttat tccaaaaccc agctttcttg tacaaagtgg ttgatctaga gggcccgcag
     1381 tggggtcagc ttctaccggg gcggcgtccc ggggtcttgg aactgcatgc agactgcctg
     1441 cttgggaaac atacttcttt atatgcccat atggacctgc taagctatgg aatgtaaaga
     1501 agtatgtatc tcaggccggg acctctctcg ccgcactgag gggcactcca caccacgggg
     1561 gccgccgcgg ccgacagcca gccaggcggc tgtggtgggg gtgaggctgg ggcggcaggc
     1621 tgcctgggcg ttggtggacg gtcgtgtccc cgccagcatt gttctgtcac tttgcctgtg
     1681 tcagcaggtg gaagtgctca ccagacattc gaaggtaagc ctatccctaa ccctctcctc
     1741 ggtctcgatt ctacgcgtac cggttagtaa tgagtttaaa cgggggaggc taactgaaac
     1801 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     1861 aacgcacggg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     1921 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     1981 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     2041 gccctgccat agcagatctg cgcagctggg gctctagggg gtatccccac gcgccctgta
     2101 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2161 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2221 ttccccgtca agctctaaat cggggcatcc ctttagggtt ccgatttagt gctttacggc
     2281 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2341 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2401 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgg
     2461 ggatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     2521 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     2581 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     2641 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     2701 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     2761 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     2821 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     2881 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     2941 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3001 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3061 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     3121 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3181 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3241 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     3301 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     3361 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     3421 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     3481 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     3541 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     3601 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     3661 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     3721 agcgggactc tggggttcgc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     3781 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     3841 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     3901 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3961 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     4021 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     4081 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4141 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4201 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4261 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4321 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4381 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4441 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4501 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4561 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4621 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt
     4681 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4741 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4801 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4861 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     4921 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4981 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5041 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5101 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5161 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5221 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5281 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5341 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5401 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5461 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5521 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5581 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5641 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5701 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5761 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5821 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5881 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5941 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6001 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6061 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6121 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6181 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
