LOCUS       sequence-43812-e        6290 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 6290)
  AUTHORS   Naughton BJ, Han DD, Gu HH
  TITLE     Fluorescence-based evaluation of shRNA efficacy.
  JOURNAL   Anal Biochem. 2011 Oct 1;417(1):162-4. Epub 2011 Jun 13.
  PUBMED    21726522
REFERENCE   2  (bases 1 to 6290)
  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"; journalName: "Anal 
            Biochem."; date: "2011-10-1"; volume: "417(1)"; pages: "162-4. Epub 
            2011 Jun 13"
COMMENT     Sequence Label: pGFPns-reporter
FEATURES             Location/Qualifiers
     source          1..6290
                     /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"
     CDS             join(900..902,903..905,906..1616,1617..1622,1623..1628,
                     1629..1739)
                     /codon_start=1
                     /product="EGFP destabilized by fusion to a mutated version 
                     of residues 422-461 of mouse ornithine decarboxylase, 
                     giving an in vivo half-life of ~1 hour"
                     /label=d1EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKSHGFPPAVAAQDDGTLPMSCAQESGMDRHPAACASARINV"
     primer_bind     complement(945..966)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1206..1225)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1553..1574
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1842..1860)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1843..1860)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1880..1897)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1886..2110
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2156..2584
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2243..2262)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2453..2474
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2593..2613)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2598..2928
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2779..2914
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2841..2860
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2995..3789
                     /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(3049..3068)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3659..3678
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3963..4084
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4000..4019)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4054..4073
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4133..4149)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4133..4149)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4146..4168)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4157..4173
                     /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(4181..4187,4188..4205,4206..4211))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4226..4247
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4364..4381)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4535..5123)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4615..4634)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5294..6085,6086..6154))
                     /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     5917..5936
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6155..6259)
                     /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 gcttccacca
      901 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
      961 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     1021 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     1081 tcgtgaccac cctgagctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     1141 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     1201 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     1261 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     1321 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     1381 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     1441 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     1501 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     1561 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagagcc
     1621 atggcttccc gccggcggtg gcggcgcagg atgatggcac gctgcccatg tcttgtgccc
     1681 aggagagcgg gatggaccgt caccctgcag cctgtgcttc tgctaggatc aatgtgtagg
     1741 taccgagctc ggatccacta gtaacggccg ccagtgtgct ggaattctgc agatatccat
     1801 cacactggcg gccgctcgag catgcatcta gagggcccta ttctatagtg tcacctaaat
     1861 gctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     1921 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     1981 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2041 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     2101 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     2161 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2221 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2281 gccggctttc cccgtcaagc tctaaatcgg ggcatccctt tagggttccg atttagtgct
     2341 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2401 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2461 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2521 attttgggga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2581 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccaggca
     2641 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     2701 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     2761 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     2821 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     2881 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     2941 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     3001 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     3061 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     3121 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     3181 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     3241 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     3301 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     3361 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     3421 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     3481 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     3541 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     3601 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     3661 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     3721 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     3781 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     3841 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     3901 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     3961 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4021 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4081 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4141 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4201 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4261 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4321 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4381 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4441 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4501 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4561 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4621 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4681 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct
     4741 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4801 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4861 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4921 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     4981 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5041 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5101 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5161 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5221 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5281 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5341 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5401 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5461 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5521 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5581 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5641 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5701 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5761 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5821 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5881 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5941 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6001 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6061 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6121 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6181 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6241 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
