LOCUS       sequence-180321-        7104 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a cGFP mRNA that ends in the MALAT1 triple helix when the 
            upstream SV40 poly(A) signal fails to be used.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7104)
  AUTHORS   Wilusz
  TITLE     Transcription Termination Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7104)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCRII-TOPO CMV-cGFP-SV40 pA + mMALAT1_3'
FEATURES             Location/Qualifiers
     source          1..7104
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     212..229
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    346..367
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(382..387,388..405,406..412)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    420..436
                     /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     425..447
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     444..460
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     444..460
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        478..496
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     478..495
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        1314..1692
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1693..1896
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1847..1867
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1940..2659
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MASKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     polyA_signal    2668..2789
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2705..2724)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2759..2778
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3776..3795)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3777..3795)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3802..3819)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3802..3818)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3811..3833)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3959..4387
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4046..4065)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4256..4277
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             4731..5525
                     /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(4785..4804)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5395..5414
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(5543..5611,5612..6403)
                     /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(5761..5780)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6574..7104,1..58)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7063..7082
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
       61 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
      121 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
      181 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      241 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      301 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      361 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      421 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagctatt
      481 taggtgacac tatagaatac tcaagctatg catcaagctt ctttaaaagg ccacttgaac
      541 tcgctttcca tggcgatttg ccttgtgagc actttcagga gagcctggaa gctgaaaaac
      601 ggtagaaaaa tttccgtgcg ggccgtgggg ggctggcggc aactgggggg ccgcagatca
      661 gagtgggcca ctggcagcca acggcccccg gggctcaggc ggggagcagc tctgtggtgt
      721 gggattgagg cgttttccaa gagtgggttt tcacgtttct aagatttccc aagcagacag
      781 cccgtgctgc tccgatttct cgaacaaaaa agcaaaacgt gtggctgtct tgggagcaag
      841 tcgcaggact gcaagcagtt gggggagaaa gtccgccatt ttgccacttc tcaaccgtcc
      901 ctgcaaggct ggggctcagt tgcgtaatgg aaagtaaagc cctgaactat cacactttaa
      961 tcttccttca aaaggtggta aactatacct actgtccctc aagagaacac aagaagtgct
     1021 ttaagaggta ttttaaaagt tccgggggtt ttgtgaggtg tttgatgacc cgtttaaaat
     1081 atgatttcca tgtttctttt gtctaaagtt tgcagctcaa atctttccac acgctagtaa
     1141 tttaagtatt tctgcatgtg tagtttgcat tcaagttcca taagctgtta agaaaaatct
     1201 agaaaagtaa aactagaacc tatttttaac cgaagaacta ctttttgcct ccctcacaaa
     1261 ggcggcggaa ggtgatcgaa ttccggtgat gcgagttgtt ctccgtcgga tccacattga
     1321 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     1381 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     1441 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     1501 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     1561 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     1621 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     1681 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     1741 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     1801 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     1861 aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact
     1921 gcttactggc gatatcacca tggccagcaa gggcgccgag ctgttcaccg gcatcgtgcc
     1981 catcctgatc gagctgaatg gcgatgtgaa tggccacaag ttcagcgtga gcggcgaggg
     2041 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     2101 gcctgtgccc tggcccaccc tggtgaccac cctgagctac ggcgtgcagt gcttctcacg
     2161 ctaccccgat cacatgaagc agcacgactt cttcaagagc gccatgcctg agggctacat
     2221 ccaggagcgc accatcttct tcgaggatga cggcaactac aagtcgcgcg ccgaggtgaa
     2281 gttcgagggc gataccctgg tgaatcgcat cgagctgacc ggcaccgatt tcaaggagga
     2341 tggcaacatc ctgggcaata agatggagta caactacaac gcccacaatg tgtacatcat
     2401 gaccgacaag gccaagaatg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     2461 tggcagcgtg cagctggccg accactacca gcagaatacc cccatcggcg atggccctgt
     2521 gctgctgccc gataaccact acctgtccac ccagagcgcc ctgtccaagg accccaacga
     2581 gaagcgcgat cacatgatct acttcggctt cgtgaccgcc gccgccatca cccacggcat
     2641 ggatgagctg tacaagtgag cggccgcaac ttgtttattg cagcttataa tggttacaaa
     2701 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2761 ggtttgtcca aactcatcaa tgtatcttag cggccgcgct gtgcggtagg cattgaggca
     2821 gccagcgcag gggcttctgc tgagggggca ggcggagctt gaggaaaccg cagataagtt
     2881 tttttctctt tgaaagatag agattaatac aactacttaa aaaatatagt caataggtta
     2941 ctaagatatt gcttagcgat tcgtcagtag ggttgtaaag gtttttcttt tcctgagaaa
     3001 acaacctttt gttttctcag gttttgcttt ttggcctttc cctagcttta aaaaaaaaaa
     3061 agcaaaagac gctggtggct ggcactcctg gtttccagga cggggttcaa gtccctgcgg
     3121 tgtctttgct tcttaagatt ttaagagaaa atatgaagac ttagaagagt agcatgagga
     3181 aggaaaagat aaaaggtttc taaaacatga cggaggttga gatgaagctt cttcatggag
     3241 taaaaaatgt atttaaaaga aaattgagag aaaggactac agagccccga attaatacca
     3301 atagaagggc aatgctttta gattaaaatg aaggtgactt aaacagctta aagtttagtt
     3361 taaaagttgt aggtgattaa aataatttga aggcgatctt ttaaaaagag attaaaccga
     3421 aggtgattaa aagaccttga aatccatgac gcagggagaa ttgcgtcatt taaagcctag
     3481 ttaacgcatt tactaaacgc agacgaaaat ggaaagatta attgggagtg gtaggatgaa
     3541 acaatttgga gaagatagaa gtttgaagtg gaaaactgga agacagaagt acgggaaggc
     3601 gaagaaaaga atagagaaga tagggaaatt agaagataaa aacatacttt tagaagaaaa
     3661 aagataaatt taaacctgaa aagtaggaag cagaagaaaa aagacaagct aggaaacaaa
     3721 aagctaaggg caaaatgtac aaactctcga gcatgcatct agagggccca attcgcccta
     3781 tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     3841 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     3901 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggac
     3961 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4021 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4081 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4141 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     4201 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4261 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     4321 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4381 gcgaatttta acaaaattca gggcgcaagg gctgctaaag gaagcggaac acgtagaaag
     4441 ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag ctactgggct atctggacaa
     4501 gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag tgggcttaca tggcgatagc
     4561 tagactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg gcgccctctg
     4621 gtaaggttgg gaagccctgc aaagtaaact ggatggcttt cttgccgcca aggatctgat
     4681 ggcgcagggg atcaagatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     4741 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     4801 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     4861 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     4921 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     4981 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     5041 catcccacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     5101 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     5161 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     5221 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     5281 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     5341 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     5401 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     5461 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     5521 tctgaattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5581 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5641 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5701 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     5761 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     5821 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     5881 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5941 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     6001 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6061 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     6121 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6181 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     6241 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     6301 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6361 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     6421 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6481 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6541 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6601 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6661 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6721 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6781 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6841 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6901 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6961 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     7021 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     7081 ttatagtcct gtcgggtttc gcca
//
