LOCUS       sequence-263350-        7116 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 7116)
  AUTHORS   Horst D, Chen J, Morikawa T, Ogino S, Kirchner T, Shivdasani RA
  TITLE     Differential WNT activity in colorectal cancer confers limited 
            tumorigenic potential and is regulated by MAPK signaling.
  JOURNAL   Cancer Res. 2012 Feb 8.
  PUBMED    22318865
REFERENCE   2  (bases 1 to 7116)
  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: "Cancer Res.
            2012 Feb 8."
COMMENT     Sequence Label: TOP-GFP
FEATURES             Location/Qualifiers
     source          1..7116
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        8..234
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             235..415
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    462..587
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1080..1313
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1498..1542
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1809..1926
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2132..2163
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      2226..2235
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2232..2234,2235..2237,2238..2951)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2232..2234,2235..2237,2238..2948)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2277..2298)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2538..2557)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2885..2906
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2967..3555
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3020..3040)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3438..3449)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3642..3875
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3947..4068
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3984..4003)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4038..4057
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4108..4243
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4263..4282)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4264..4282)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4292..4309)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4292..4308)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4301..4323)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4450..4905
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4537..4556)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4747..4768
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4931..5035
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5036..5104,5105..5896)
                     /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(5254..5273)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6067..6655
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6556..6575
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6809..6826
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6943..6964
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6979..6984,6985..7002,7003..7009)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7017..7033
                     /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     7022..7044
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7041..7057
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7041..7057
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7076..7096
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7078..7096
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag
       61 caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg
      121 tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact
      181 gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct
      241 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      301 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      361 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
      421 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
      481 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
      541 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
      601 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
      661 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
      721 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
      781 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
      841 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
      901 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
      961 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1021 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1081 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1141 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1201 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1261 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1321 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1381 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     1441 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     1501 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     1561 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     1621 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     1681 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     1741 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat
     1801 cggttaactt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     1861 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     1921 aattttatcg atcacgagac tagcctcgag aagcttgata tcacgacgcg tggtaccgag
     1981 ctcttacgcg agatcaaagg gggtaagatc aaagggggta agatcaaagg ggcgcgagat
     2041 caaagggggt aagatcaaag ggggtaagat caaagggggt aagatcaaag gggcgcgccc
     2101 gcgtgctagc ccgggctcga gatctagact ctagagggta tataatggaa gctcgaattc
     2161 cagcttggca ttccggtact gttggtaaaa agcttggcat tccggtactg ttggatccac
     2221 cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     2281 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     2341 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     2401 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     2461 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     2521 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     2581 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     2641 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     2701 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     2761 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     2821 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     2881 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     2941 tgtacaagta aagcggccgc gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat
     3001 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     3061 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     3121 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     3181 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     3241 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     3301 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     3361 agctgacgtc ctttccatgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     3421 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     3481 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     3541 gggccgcctc cccgcctgga attcgagctc ggtaccttta agaccaatga cttacaaggc
     3601 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     3661 ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg
     3721 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     3781 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     3841 cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat
     3901 tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc
     3961 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4021 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct
     4081 ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     4141 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     4201 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt
     4261 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact
     4321 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     4381 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     4441 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4501 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     4561 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4621 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     4681 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4741 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     4801 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     4861 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     4921 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4981 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5041 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5101 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5161 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5221 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5281 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5341 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5401 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5461 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5521 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     5581 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     5641 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     5701 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     5761 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     5821 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     5881 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     5941 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6001 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6061 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6121 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6181 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6241 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6301 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6361 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6421 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6481 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6541 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6601 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6661 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     6721 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     6781 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     6841 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     6901 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     6961 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     7021 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat
     7081 taaccctcac taaagggaac aaaagctgga gctgca
//
