LOCUS       sequence-251063-        5952 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 5952)
  AUTHORS   Ungar AR, Moon RT
  TITLE     Inhibition of protein kinase A phenocopies ectopic expression of 
            hedgehog in the CNS of wild-type and cyclops mutant embryos.
  JOURNAL   Dev Biol. 1996 Aug 25;178(1):186-91. doi: 10.1006/dbio.1996.0209.
  PUBMED    8812120
REFERENCE   2  (bases 1 to 5952)
  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: "10.1006/dbio.1996";
            journalName: "Dev Biol"; date: "1996-08-25- 25"; volume: "178"; 
            issue: "1"; pages: "186-91"
COMMENT     Sequence Label: M7 pdnPKA-GFP
FEATURES             Location/Qualifiers
     source          1..5952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        785..803
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     785..802
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1987..2700
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2017..2045)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2640..2660
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    2754..2888
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2778..2797)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2832..2851
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3001..3021)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3001..3019)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3040..3056)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3040..3056)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3053..3075)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3064..3080
                     /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(3088..3094,3095..3112,3113..3118))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3133..3154
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3271..3288)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3442..4030)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3522..3541)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4201..4992,4993..5061))
                     /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     4824..4843
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5062..5166)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5192..5647)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5329..5350)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5541..5560
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(5718..5952,1..750)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 gtcttggccc tgtgccaagt ccgccatatt gaattggcat ggtgccaata atggcggcca
       61 tattggctat atgccaggat caatatatag gcaatatcca atatggccct atgccaatat
      121 ggctattggc caggttcaat actatgtatt ggccctatgc catatagtat tccatatatg
      181 ggttttccta ttgacgtaga tagcccctcc caatgggcgg tcccatatac catatatggg
      241 gcttcctaat accgcccata gccactcccc cattgacgtc aatggtctct atatatggtc
      301 tttcctattg acgtcatatg ggcggtccta ttgacgtata tggcgcctcc cccattgacg
      361 tcaattacgg taaatggccc gcctggctca atgcccattg acgtcaatag gaccacccac
      421 cattgacgtc aatgggatgg ctcattgccc attcatatcc gttctcacgc cccctattga
      481 cgtcaatgac ggtaaatggc ccacttggca gtacatcaat atctattaat agtaacttgg
      541 caagtacatt actattggaa gtacgccagg gtacattggc agtactccca ttgacgtcaa
      601 tggcggtaaa tggcccgcga tggctgccaa gtacatcccc attgacgtca atggggaggg
      661 gcaatgacgc aaatgggcgt tccattgacg taaatgggcg gtaggcgtgc ctaatgggag
      721 gtctatataa gcaatgctcg tttagggaac cgccattctg cctggggacg tcggagcaag
      781 cttgatttag gtgacactat agaatacaag ctacttgttc tttttgcagg atccatggcg
      841 tctggcagta tggcaaccag tgaggaagag cggagtctcc gggaatgcga gctctatgtg
      901 cagaagcaca atatccaggc cctgctgaag gactccatcg tgcagctgtg cactacgcgg
      961 cccgagaggc ccatggcatt ccttcgggaa tactttgaga ggttggagaa ggaggaggca
     1021 agacagattc agtgtctaca gaaaaccggc atccgtactg actcgaggga ggacgagatc
     1081 tctcctccac cccccaatcc agtggtgaag ggccgacggc gccgaggtgc tatcagtgct
     1141 gaagtttaca ctgaggagga tgctgcctcc tacgttagaa aggttattcc aaaagattat
     1201 aagacaatgg ctgctttagc caaggccatc gaaaagaatg tgctgttttc acaccttgat
     1261 gataacgaga gaagtgacat ttttgatgct atgtttccag tctcctttat tgctggagag
     1321 acggttattc agcaaggtga tgaaggggat aacttctatg tgatcgatca aggagaaatg
     1381 gatgtctatg tcaataatga atgggcaacc agtgttgggg aaggagggag ctttgaagag
     1441 ctggctttga tttatggaac acccagagca gccactgtca aagcaaagac aaacgtgaaa
     1501 ctgtggggca tcgaccgaga cagctaccga agaatcctca tgggaagcac tctgcgaaag
     1561 aggaagatgt atgaagaatt ccttagtaaa gtgtctattt tagagtctct ggacaagtgg
     1621 gagcgtctca cagtagccga tgcattggag cctgtccagt ttgaagatgg acagaagatc
     1681 gtggtgcaag gagagccggg ggatgagttc ttcatcattt tagagggcac agctgctgtg
     1741 ctgcagcgtc ggtcagaaaa cgaagaattt gttgaagtgg gacgactggg gccttctgat
     1801 tattttgatg aaattgccct gctgatgaat catcctcggg ctgccactgt ggttgcccgg
     1861 ggccctttga agtgcgttaa gttggaccgg cctcggtttg aacgcgtcct tggcccgtgc
     1921 tcagacatcc tcaagcggaa catccagcag tacaacagct tcgtgtccct gtccgtccat
     1981 cgattgatga gtaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta
     2041 gatggtgatg ttaatgggca caaattttct gtcagtggag agggtgaagg tgatgcaaca
     2101 tacggaaaac ttacccttaa atttatttgc actactggaa aactacctgt tccatggcca
     2161 acacttgtca ctactttcac ttatggtgtt caatgctttt caagataccc agatcatatg
     2221 aaacggcatg actttttcaa gagtgccatg cccgaaggtt atgtacagga aagaactata
     2281 tttttcaaag atgacgggaa ctacaagaca cgtgctgaag tcaagtttga aggtgatacc
     2341 cttgttaata gaatcgagtt aaaaggtatt gattttaaag aagatggaaa cattcttgga
     2401 cacaaattgg aatacaacta taactcacac aatgtataca tcatggcaga caaacaaaag
     2461 aatggaatca aagttaactt caaaattaga cacaacattg aagatggaag cgttcaacta
     2521 gcagaccatt atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt accagacaac
     2581 cattacctgt ccacacaatc tgccctttcg aaagatccca acgaaaagag agaccacatg
     2641 gtccttcttg agtttgtaac agctgctggg attacacatg gcatggatga actatacaaa
     2701 taagaattca aggcctctcg agcctctaga actatagtga gtcgtattac gtagatccag
     2761 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     2821 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     2881 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     2941 aggtttttta attcgcggcc gcggcgccaa tgcattgggc ccggtaccca gcttttgttc
     3001 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     3061 aaattgttat ccgctcacaa ttccacacaa catacgagcc gggagcataa agtgtaaagc
     3121 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     3181 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     3241 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     3301 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     3361 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     3421 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3481 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     3541 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     3601 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     3661 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     3721 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     3781 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     3841 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     3901 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     3961 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4021 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4081 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     4141 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     4201 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     4261 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     4321 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     4381 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     4441 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     4501 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     4561 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     4621 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     4681 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     4741 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     4801 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     4861 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     4921 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     4981 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     5041 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     5101 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     5161 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa
     5221 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     5281 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     5341 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     5401 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     5461 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     5521 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     5581 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     5641 ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     5701 tcttcgctat tacgccagtc gaccgccaat tcaatatggc gtatatggac tcatgccaat
     5761 tcaatatggt ggatctggac ctgtgccaat tcaatatggc gtatatggac tcgtgccaat
     5821 tcaatatggt ggatctggac cccagccaat tcaatatggc ggacttggca ccatgccaat
     5881 tcaatatggc ggacctggca ctgtgccaac tggggagggg tctacttggc acggtgccaa
     5941 gtttgaggag gg
//
