LOCUS       sequence-66024-e        6220 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 6220)
  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 6220)
  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-235
FEATURES             Location/Qualifiers
     source          1..6220
                     /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(1333..1357)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1710..1751
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1791..1810)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1835..1883
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2085..2513
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2172..2191)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2382..2403
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2522..2542)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2527..2856
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2707..2842
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2769..2788
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2923..3717
                     /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(2977..2996)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3587..3606
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3893..4014
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3930..3949)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3984..4003
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4063..4079)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4063..4079)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4076..4098)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4087..4103
                     /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(4111..4117,4118..4135,4136..4141))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4156..4177
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4294..4311)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4465..5053)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4545..4564)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5224..6015,6016..6084))
                     /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     5847..5866
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6085..6189)
                     /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 aggctggacc ttcaattggt accaatcgaa
      961 actcgttttc ttctaagtgt atgtatgtgt gtcctttctt cagatgacac cacctgtgga
     1021 tggttcttaa ttttctatct attccttatt cttccaagtg ctatctattc gtttgctgaa
     1081 aatcgtccga agatatcagg atcaggcctt ggctgattgc aaaattgttc accgtgaaaa
     1141 ttaaatattg cactctcccc ggcctgatct gagagtaagg cgaagctgaa ttgactttaa
     1201 ttcgacaggt ttgtttttgc ttcaattgtt taaaattttt aaatgggttt tagttttagc
     1261 aggtttcttt atatactctg tggaattttt taaaagtatc tgttgtgtta taatgtgcta
     1321 atagaaaaca aaacccagct ttcttgtaca aagtggttga tctagagggc ccgcagtggg
     1381 gtcagcttct accggggcgg cgtcccgggg tcttggaact gcatgcagac tgcctgcttg
     1441 ggaaacatac ttctttatat gcccatatgg acctgctaag ctatggaatg taaagaagta
     1501 tgtatctcag gccgggacct ctctcgccgc actgaggggc actccacacc acgggggccg
     1561 ccgcggccga cagccagcca ggcggctgtg gtgggggtga ggctggggcg gcaggctgcc
     1621 tgggcgttgg tggacggtcg tgtccccgcc agcattgttc tgtcactttg cctgtgtcag
     1681 caggtggaag tgctcaccag acattcgaag gtaagcctat ccctaaccct ctcctcggtc
     1741 tcgattctac gcgtaccggt tagtaatgag tttaaacggg ggaggctaac tgaaacacgg
     1801 aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg
     1861 cacgggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     1921 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     1981 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     2041 tgccatagca gatctgcgca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     2101 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2161 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2221 ccgtcaagct ctaaatcggg gcatcccttt agggttccga tttagtgctt tacggcacct
     2281 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2341 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2401 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttggggat
     2461 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     2521 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2581 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     2641 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     2701 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     2761 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     2821 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     2881 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     2941 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     3001 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     3061 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     3121 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     3181 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     3241 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     3301 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     3361 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     3421 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     3481 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     3541 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     3601 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     3661 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     3721 ggactctggg gttcgcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     3781 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     3841 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     3901 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     3961 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4021 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     4081 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4141 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4201 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4261 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4321 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4381 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4441 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4501 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4561 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4621 gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct
     4681 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4741 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4801 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4861 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     4921 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4981 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5041 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5101 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5161 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5221 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5281 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5341 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5401 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5461 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5521 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5581 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5641 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5701 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5761 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5821 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5881 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5941 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6001 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6061 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6121 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6181 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
