LOCUS       sequence-159487-        7146 bp    DNA     circular SYN 14-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7146)
  AUTHORS   Chen JK, Taipale J, Cooper MK, Beachy PA
  TITLE     Inhibition of Hedgehog signaling by direct binding of cyclopamine to
            Smoothened.
  JOURNAL   Genes Dev. 2002 Nov 1. 16(21):2743-8.
  PUBMED    12414725
REFERENCE   2  (bases 1 to 7146)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Genes Dev. 
            2002 Nov 1. 16(21):2743-8."
COMMENT     Sequence Label: pEGFP-mSmo
FEATURES             Location/Qualifiers
     source          1..7146
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        16..319
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        320..523
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     474..494
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2963..2972
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2969..2971,2972..2974,2975..3685)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2969..2971,2972..2974,2975..3685)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3014..3035)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3275..3294)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3622..3643
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3889..4010
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3926..3945)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3980..3999
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4017..4472)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4154..4175)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4366..4385
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4499..4603
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4605..4962
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4605..4796
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4628..4648)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4813..4948
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4997..5791
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4997..5791
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5051..5070)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5661..5680
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5979..5998)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6023..6070
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6399..6987
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6888..6907
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
       61 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      121 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      181 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      241 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      301 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      361 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      421 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      481 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
      541 gatccgctac aagcttacca tggccgctgg ccgccccgtg cgtgggcccg agctggcgcc
      601 ccggaggctg ctgcagttgc tgctgctggt actgctgggg ggcccgggcc ggggggcggc
      661 cttgagcggg aacgtgaccg ggcctgggcc tcacagcgcc agcgggagct cgaggaggaa
      721 cgtgccggtg accagccctc cgccgccgct gctgagccac tgcggccggg ccgcccactg
      781 cgagcctttg cgctacaacg tgtgcctggg ctcggcgctg ccctacggag ccaccaccac
      841 gctgctggct ggggactcgg actcgcagga ggaagcgcac ggcaagctcg tgctctggtc
      901 cggcctccgg aatgcccccc gctgctgggc agtgatccag cccctgctgt gtgctgtcta
      961 catgcccaag tgtgagaatg accgagtgga gttgcccagc cgtaccctct gccaggccac
     1021 ccgaggcccc tgtgccattg tggagcggga gcgagggtgg cctgactttc tgcgttgcac
     1081 accggaccac ttccctgaag gctgcccaaa cgaggtacaa aacatcaagt tcaacagctc
     1141 aggccaatgt gaagcaccct tggtgcgaac agacaacccc aagagctggt atgaggacgt
     1201 ggagggctgt gggattcagt gtcagaaccc gctgttcacc gaggccgagc accaggacat
     1261 gcacagctac atcgcagcct tcggggcggt cactggtctc tgcacgctct tcacactggc
     1321 cacctttgtg gctgactggc ggaattccaa tcgctaccct gcggttattc tcttctatgt
     1381 caatgcgtgt ttcttcgtgg gcagcattgg ctggctggcc cagttcatgg atggtgcccg
     1441 ccgagagatt gtttgccgag cagatggcac catgagattt ggggagccca cctccagtga
     1501 gaccctgtcc tgtgtcatca tctttgtcat tgtgtactat gccttgatgg ctggagtagt
     1561 ctggttcgtg gtcctcacct atgcctggca cacctccttc aaagccctgg gcaccaccta
     1621 ccagcctctc tcgggcaaga catcctattt ccacctgctc acgtggtcac tcccctttgt
     1681 cctcacggtg gcaatcctgg ctgtggctca ggtagatgga gactccgtga gtggcatctg
     1741 ttttgtaggc tacaagaact atcggtaccg tgctggcttt gtcctggccc caattggcct
     1801 ggtgcttatt gtgggaggct acttcctcat cagaggggtc atgactctgt tctccatcaa
     1861 gagcaaccac cctgggctct taagtgagaa ggcagccagc aagatcaacg agaccatgct
     1921 gcgcctgggc atttttggct tcctggcctt tggctttgtg ctcatcacct tcagctgcca
     1981 cttctatgac ttcttcaacc aggctgagtg ggagcgtagc ttccgggact atgtgctatg
     2041 ccaagccaac gtgaccatcg ggctgcctac caagaagccc attcctgact gtgagatcaa
     2101 gaatcggccc agcctcctgg tggagaagat caatctattt gccatgtttg gcactggcat
     2161 tgccatgagc acctgggtct ggaccaaggc caccctgctc atctggaggc gcacctggtg
     2221 caggttgact gggcacagtg atgatgagcc caagagaatc aagaagagca agatgatcgc
     2281 caaggccttc tctaagcggc gtgagctgct gcagaacccg ggccaggagc tctccttcag
     2341 catgcacact gtctcccatg atggacctgt tgcgggtttg gcttttgacc tcaatgaacc
     2401 ctcagctgac gtctcctctg cctgggctca gcatgtcacc aagatggtgg ctcggagagg
     2461 agccatattg ccccaggatg tgtccgttac ccctgtggca actccagtgc caccagaaga
     2521 acaagccaac atgtggctgg ttgaggcaga gatctcccca gagttagaga agcgtttggg
     2581 ccggaagaaa aagcggagga agaggaagaa ggaggtgtgc cccttggggc cagcccctga
     2641 gcttcaccac tctgcccctg ttcctgccac cagtgcagtt cctcggctgc ctcagctgcc
     2701 tcggcagaag tgcctggtag ctgcaaacgc ctggggaaca ggggagtcct gccgacaggg
     2761 agcctggact ctagtctcca accccttctg cccagagcct agtccccatc aagatccatt
     2821 tctccctggt gcctcagccc cccgggtctg ggctcagggc cgcctccagg ggctgggatc
     2881 cattcattcc cgcactaacc taatggaggc tgagatcttg gatgcagact cggacttcag
     2941 aaaagagcta gcgctaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
     3001 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     3061 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     3121 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     3181 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     3241 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     3301 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     3361 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     3421 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     3481 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     3541 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     3601 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     3661 tctcggcatg gacgagctgt acaagtccgg actcagatct cgagctcaag cttcgaattc
     3721 tgcaggaatt aggtcgtagt cgacggtacc gcgggcccgg gatccaccgg atctagataa
     3781 ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     3841 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     3901 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     3961 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta acgcgtaaat
     4021 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     4081 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     4141 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     4201 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     4261 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     4321 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     4381 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     4441 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     4501 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4561 ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac
     4621 cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     4681 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     4741 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     4801 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4861 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     4921 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
     4981 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     5041 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     5101 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     5161 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     5221 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     5281 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     5341 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     5401 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     5461 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     5521 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     5581 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     5641 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     5701 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     5761 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     5821 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     5881 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     5941 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     6001 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     6061 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     6121 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     6181 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     6241 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6301 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6361 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6421 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6481 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6541 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6601 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6661 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6721 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6781 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6841 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6901 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6961 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7021 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     7081 aaccgtatta ccgccatgca ttagttatta atagtaatca attacggggt cattagttca
     7141 tagccc
//
