LOCUS       sequence-66034-e        6209 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 6209)
  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 6209)
  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 hsa-let-7a-1
FEATURES             Location/Qualifiers
     source          1..6209
                     /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(1322..1346)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1699..1740
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1780..1799)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1824..1872
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2074..2502
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2161..2180)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2371..2392
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2511..2531)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2516..2845
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2696..2831
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2758..2777
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2912..3706
                     /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(2966..2985)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3576..3595
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3882..4003
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3919..3938)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3973..3992
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4052..4068)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4052..4068)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4065..4087)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4076..4092
                     /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(4100..4106,4107..4124,4125..4130))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4145..4166
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4283..4300)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4454..5042)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4534..4553)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5213..6004,6005..6073))
                     /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     5836..5855
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6074..6178)
                     /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 aggctcattt tcaacgtaag tgaatgaaaa
      961 tggtctaata ctatttttct tatcactcac acaggaaacc aggattaccg aggaggaaaa
     1021 aaagccttcc tgtggtgctc aactgtgatt ccttttcacc attcaccctg gatgttctct
     1081 tcactgtggg atgaggtagt aggttgtata gttttagggt cacacccacc actgggagat
     1141 aactatacaa tctactgtct ttcctaacgt gatagaaaag tctgcatcca ggcggtctga
     1201 tagaaagtca gttaactaat tgtacaatat ttaagattaa cttgtcttaa agagatgtag
     1261 tgcagcattt gtttatggcc tggaaataaa ttaatttaga gataaagtct gtagcaagta
     1321 cacccagctt tcttgtacaa agtggttgat ctagagggcc cgcagtgggg tcagcttcta
     1381 ccggggcggc gtcccggggt cttggaactg catgcagact gcctgcttgg gaaacatact
     1441 tctttatatg cccatatgga cctgctaagc tatggaatgt aaagaagtat gtatctcagg
     1501 ccgggacctc tctcgccgca ctgaggggca ctccacacca cgggggccgc cgcggccgac
     1561 agccagccag gcggctgtgg tgggggtgag gctggggcgg caggctgcct gggcgttggt
     1621 ggacggtcgt gtccccgcca gcattgttct gtcactttgc ctgtgtcagc aggtggaagt
     1681 gctcaccaga cattcgaagg taagcctatc cctaaccctc tcctcggtct cgattctacg
     1741 cgtaccggtt agtaatgagt ttaaacgggg gaggctaact gaaacacgga aggagacaat
     1801 accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc acgggtgttg
     1861 ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac
     1921 cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa
     1981 gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcag
     2041 atctgcgcag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     2101 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2161 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2221 taaatcgggg catcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2281 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2341 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2401 tcaaccctat ctcggtctat tcttttgatt tataagggat tttggggatt tcggcctatt
     2461 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     2521 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2581 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2641 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     2701 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     2761 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     2821 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     2881 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     2941 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     3001 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     3061 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     3121 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     3181 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     3241 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     3301 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     3361 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     3421 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     3481 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     3541 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     3601 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     3661 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     3721 ttcgcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     3781 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     3841 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     3901 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3961 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4021 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4081 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4141 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4201 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4261 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4321 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4381 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4441 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4501 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4561 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4621 tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt
     4681 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4741 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4801 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4861 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     4921 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4981 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5041 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5101 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5161 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5221 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5281 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5341 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5401 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5461 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5521 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5581 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5641 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5701 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5761 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5821 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5881 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5941 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6001 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6061 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6121 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6181 catttccccg aaaagtgcca cctgacgtc
//
