LOCUS       sequence-66681-e        6954 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express cGFP transcript ending in a poly(A) tail [using the SV40 
            poly(A) signal].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6954)
  AUTHORS   Wilusz JE, JnBaptiste CK, Lu LY, Kuhn CD, Joshua-Tor L, Sharp PA
  TITLE     A triple helix stabilizes the 3' ends of long noncoding RNAs that 
            lack poly(A) tails.
  JOURNAL   Genes Dev. 2012 Nov 1;26(21):2392-407. doi: 10.1101/gad.204438.112. 
            Epub 2012 Oct 16.
  PUBMED    23073843
REFERENCE   2  (bases 1 to 6954)
  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: 
            "10.1101/gad.204438.112"; journalName: "Genes Dev"; date: 
            "2012-11-1- 1"; volume: "26"; issue: "21"; pages: "2392-407"
COMMENT     Sequence Label: pCRII-TOPO CMV-cGFP-SV40 Poly(A) Sense
FEATURES             Location/Qualifiers
     source          1..6954
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        1075..1453
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1454..1657
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1608..1628
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     polyA_signal    2426..2547
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2463..2482)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2517..2536
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3387..3406)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3388..3406)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3413..3430)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3413..3429)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3422..3444)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3570..3998
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3657..3676)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3867..3888
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             4342..5136
                     /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(4396..4415)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5006..5025
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(5154..5222,5223..6014)
                     /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     complement(5372..5391)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6185..6773
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6674..6693
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6927..6944
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tctttaaaag gccacttgaa
      301 ctcgctttcc atggcgattt gccttgtgag cactttcagg agagcctgga agctgaaaaa
      361 cggtagaaaa atttccgtgc gggccgtggg gggctggcgg caactggggg gccgcagatc
      421 agagtgggcc actggcagcc aacggccccc ggggctcagg cggggagcag ctctgtggtg
      481 tgggattgag gcgttttcca agagtgggtt ttcacgtttc taagatttcc caagcagaca
      541 gcccgtgctg ctccgatttc tcgaacaaaa aagcaaaacg tgtggctgtc ttgggagcaa
      601 gtcgcaggac tgcaagcagt tgggggagaa agtccgccat tttgccactt ctcaaccgtc
      661 cctgcaaggc tggggctcag ttgcgtaatg gaaagtaaag ccctgaacta tcacacttta
      721 atcttccttc aaaaggtggt aaactatacc tactgtccct caagagaaca caagaagtgc
      781 tttaagaggt attttaaaag ttccgggggt tttgtgaggt gtttgatgac ccgtttaaaa
      841 tatgatttcc atgtttcttt tgtctaaagt ttgcagctca aatctttcca cacgctagta
      901 atttaagtat ttctgcatgt gtagtttgca ttcaagttcc ataagctgtt aagaaaaatc
      961 tagaaaagta aaactagaac ctatttttaa ccgaagaact actttttgcc tccctcacaa
     1021 aggcggcgga aggtgatcga attccggtga tgcgagttgt tctccgtcgg atccacattg
     1081 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     1141 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     1201 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     1261 ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac atcaagtgta
     1321 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     1381 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     1441 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     1501 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     1561 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     1621 taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac
     1681 tgcttactgg cgatatcatg gccagcaagg gcgaggagct gttcaccggc gtggtgccca
     1741 tcctggtgga gctggacggc gatgtgaatg gccacaagtt cagcgtgagc ggcgagggcg
     1801 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1861 ctgtgccctg gcccaccctg gtgaccaccc tgagctacgg cgtgcagtgc ttctcacgct
     1921 accccgatca catgaagcag cacgacttct tcaagagcgc catgcctgag ggctacatcc
     1981 aggagcgcac catcttcttc gaggatgacg gcaactacaa gtcgcgcgcc gaggtgaagt
     2041 tcgagggcga taccctggtg aatcgcatcg agctgaccgg caccgatttc aaggaggatg
     2101 gcaacatcct gggcaataag atggagtaca actacaacgc ccacaatgtg tacatcatga
     2161 ccgacaaggc caagaatggc atcaaggtga acttcaagat ccgccacaac atcgaggatg
     2221 gcagcgtgca gctggccgac cactaccagc agaatacccc catcggcgat ggccctgtgc
     2281 tgctgcccga taaccactac ctgtccaccc agagcgccct gtccaaggac cccaacgaga
     2341 agcgcgatca catgatctac ttcggcttcg tgaccgccgc cggcatcacc cacggcatgg
     2401 acgagctgta caagtgagcg gccgcaactt gtttattgca gcttataatg gttacaaata
     2461 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     2521 tttgtccaaa ctcatcaatg tatcttagcg gccgcgctgt gcggtaggca ttgaggcagc
     2581 cagcgcaggg gcttctgctg agggggcagg cggagcttga ggaaaccgca gataagtttt
     2641 tttctctttg aaagatagag attaatacaa ctacttaaaa aatatagtca ataggttact
     2701 aagatattgc ttagcgttaa gtttttaacg taattttaat agcttaagat tttaagagaa
     2761 aatatgaaga cttagaagag tagcatgagg aaggaaaaga taaaaggttt ctaaaacatg
     2821 acggaggttg agatgaagct tcttcatgga gtaaaaaatg tatttaaaag aaaattgaga
     2881 gaaaggacta cagagccccg aattaatacc aatagaaggg caatgctttt agattaaaat
     2941 gaaggtgact taaacagctt aaagtttagt ttaaaagttg taggtgatta aaataatttg
     3001 aaggcgatct tttaaaaaga gattaaaccg aaggtgatta aaagaccttg aaatccatga
     3061 cgcagggaga attgcgtcat ttaaagccta gttaacgcat ttactaaacg cagacgaaaa
     3121 tggaaagatt aattgggagt ggtaggatga aacaatttgg agaagataga agtttgaagt
     3181 ggaaaactgg aagacagaag tacgggaagg cgaagaaaag aatagagaag atagggaaat
     3241 tagaagataa aaacatactt ttagaagaaa aaagataaat ttaaacctga aaagtaggaa
     3301 gcagaagaaa aaagacaagc taggaaacaa aaagctaagg gcaaaatgta caaactctcg
     3361 agcatgcatc tagagggccc aattcgccct atagtgagtc gtattacaat tcactggccg
     3421 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     3481 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     3541 aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc
     3601 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3661 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3721 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3781 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3841 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3901 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3961 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaattc agggcgcaag
     4021 ggctgctaaa ggaagcggaa cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg
     4081 atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag
     4141 gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc
     4201 gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac
     4261 tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagatc tgatcaagag
     4321 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     4381 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     4441 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     4501 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     4561 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     4621 ttgggcgaag tgccggggca ggatctcctg tcatcccacc ttgctcctgc cgagaaagta
     4681 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4741 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     4801 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4861 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4921 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4981 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5041 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     5101 atcgccttct atcgccttct tgacgagttc ttctgaattg aaaaaggaag agtatgagta
     5161 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5221 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5281 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5341 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5401 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5461 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5521 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5581 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5641 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     5701 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     5761 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     5821 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     5881 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     5941 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6001 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6061 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6121 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6181 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6241 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6301 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6361 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6421 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6481 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6541 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6601 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6661 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6721 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6781 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     6841 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     6901 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaag
//
