LOCUS       Exported                7007 bp ds-DNA     circular SYN 12-MAR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7007)
  AUTHORS   Wythe Lab
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Jul 7, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7007
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     170..189
                     /label=oJDW 855 (CMV FWD)
     primer_bind     360..391
                     /label=FWD
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     exon            662..751
                     /label=chicken B-actin exon
     intron          752..1670
                     /label=chicken b-actin intron + rabbit b-globin intron
     misc_feature    1594..1616
                     /label=pCAGGS FWDSeq
     intron          1671..1723
                     /label=rabbit B-globin intron
     primer_bind     complement(1697..1723)
                     /label=Primer 1
     primer_bind     1737..1806
                     /label=oJDW 2415
     protein_bind    1737..1761
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1803..2453
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAH"
     CDS             2508..2780
                     /codon_start=1
                     /product="degron consisting of residues 30-120 of human 
                     Cdt1 (Zielke and Edgar, 2015)"
                     /label=Cdt1 (30-120)
                     /translation="PSPARPALRAPASATSGSRKRARPPAAPGRDQARPPARRRLRLSV
                     DEVSSPSTPEAPDIPACPSPGQKIKKSTPAAGQPPHLTSAQDQDTI"
     CDS             2787..2840
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2847..3524
                     /codon_start=1
                     /product="monomeric Azami-Green fluorescent protein 
                     (Karasawa et al., 2003)"
                     /label=mAzami-Green
                     /translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
                     TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
                     TSNISMRGDCFFYDIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVEDGVLKGDVNMRLL
                     LEGGGHYRCDFKTTYKAKKEVRLPDAHKIDHRIEILKHDKDYNKVKLYENAVARYSMLP
                     SQAK"
     CDS             3555..3884
                     /codon_start=1
                     /product="degron consisting of residues 1-110 of human 
                     Geminin (Zielke and Edgar, 2015)"
                     /label=Gem1 (1-110)
                     /translation="MNPSMKQKQEEIKENIKNSSVPRRTLKMIQPSASGSLVGRENELS
                     AGLSKRKHRNDHLTSTTSSPGVIVPESSENKNLGGVTQESFDLMIKENPSSQYWKEVAE
                     KRRKAL"
     primer_bind     complement(3870..3938)
                     /label=oJDW 2416
     protein_bind    complement(3914..3938)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    4095..4150
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4509..4525)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4533..4549
                     /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(4557..4587)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4602..4623
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4681..4877
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4728..4863
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4883..5017
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    complement(4920..4939)
                     /label=SV40pA REVSeq
     rep_origin      complement(5256..5844)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6015..6875)
                     /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"
     promoter        complement(6876..6980)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcctcgag gatatcacaa
     1741 gtttgtacaa aaaagcaggc tggtctgact ggtacctcgt gattaattaa gaattgggat
     1801 ccatggtgtc cgtgatcaag cccgagatga agatgcggta ctacatggac ggcagcgtga
     1861 acggccacga gttcaccatc gagggcgagg gcaccggcag accttacgag ggccaccagg
     1921 aaatgaccct gagagtcacc atggccgagg gcggacccat gcccttcgcc ttcgacctgg
     1981 tgtcccacgt gttctgctac ggccaccggg tgttcaccaa gtaccccgag gaaatccccg
     2041 actacttcaa gcaggccttc cccgagggcc tgagctggga gcggagcctg gaattcgagg
     2101 atggcggcag cgcctctgtg tctgcccaca tcagcctgcg gggcaacacc ttctaccaca
     2161 agagcaagtt caccggcgtg aacttccccg ccgacggccc catcatgcag aaccagagcg
     2221 tggactggga gcccagcacc gagaagatca ccgccagcga cggcgtgctg aagggcgacg
     2281 tgaccatgta cctgaagctg gaaggcggcg gaaaccacaa gtgccagatg aaaaccacct
     2341 acaaggccgc caaagaaatc ctggaaatgc ccggcgacca ctacatcggc caccggctgg
     2401 tgcggaaaac cgagggcaac atcaccgagc aggtggagga tgccgtggcc cactacagca
     2461 tgctgcctag cctcggcggc taccctagcc attggaggcc cctggaaccc tctcctgcca
     2521 gacctgccct gagagcccct gcctccgcca cctccggcag cagaaagaga gccagacctc
     2581 ctgccgcccc tggcagagat caggccaggc cccctgcccg cagacggctc cggctgtccg
     2641 tggatgaggt gtccagccct agcacccccg aggcccccga tatccctgcc tgtcccagcc
     2701 ctggccagaa aatcaagaag agcacacctg ccgccggaca gcctcctcac ctgaccagcg
     2761 cccaggacca ggacaccatc gtcgacgagg gcaggggcag cctgctgacc tgcggcgacg
     2821 tggaggagaa ccccggcccc agatccatgg tgtccgtgat caagcccgag atgaagatca
     2881 agctgtgcat gcggggaacc gtgaacggcc acaacttcgt gatcgagggc gagggcaagg
     2941 gaaaccccta cgagggcacc cagatcctgg acctgaacgt gaccgaaggc gcccctctgc
     3001 ccttcgccta cgacatcctg accaccgtgt tccagtacgg caaccgggcc ttcaccaagt
     3061 accccgccga catccaggac tacttcaagc agaccttccc cgagggctac cactgggagc
     3121 ggagcatgac ctacgaggac cagggcatct gcaccgccac ctccaacatc agcatgaggg
     3181 gcgactgctt tttctacgac atcagattcg acggcaccaa cttccccccc aacggccccg
     3241 tgatgcagaa aaagaccctg aagtgggagc ccagcaccga gaagatgtac gtggaggacg
     3301 gcgtgctgaa gggcgacgtg aacatgcggc tgctgctgga aggcggcgga cactacagat
     3361 gcgacttcaa gaccacctac aaggccaaga aagaagtgcg gctgcccgac gcccacaaga
     3421 tcgaccaccg gatcgagatc ctgaagcacg acaaggacta caacaaagtg aagctgtacg
     3481 agaacgccgt ggccaggtac agcatgctgc ccagccaggc caagggctac cccagccact
     3541 ggcggcctct ggaaatgaac cccagcatga agcagaagca ggaagagatc aaagagaaca
     3601 tcaagaacag cagcgtgccc aggcggaccc tgaagatgat ccagcccagc gccagcggat
     3661 ctctggtggg ccgggagaat gagctgtctg ccggcctgag caagcggaag caccggaacg
     3721 accacctgac cagcaccacc tctagcccag gcgtgatcgt gcccgagagc agcgagaaca
     3781 agaacctggg cggcgtgacc caagaaagct tcgacctgat gatcaaagaa aaccccagca
     3841 gccagtactg gaaagaggtg gccgagaagc ggcggaaggc cctgtagaga tctgactgag
     3901 cggccgccag tcaacccagc tttcttgtac aaagtggtga tatcgcggcc gcactcctca
     3961 ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata
     4021 ccactgagat ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca
     4081 tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt
     4141 gtgtctctca ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta
     4201 tttggtttag agtttggcaa catatgccat atgctggctg ccatgaacaa aggtggctat
     4261 aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc catagaaaag
     4321 ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt tctttaacat
     4381 ccctaaaatt ttccttacat gttttactag ccagattttt cctcctctcc tgactactcc
     4441 cagtcatagc tgtccctctt ctcttatgaa gatccctcga cctgcagccc aagcttggcg
     4501 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4561 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4621 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc
     4681 cgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     4741 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     4801 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     4861 gcctaggctt ttgcaaaaag ctaacttgtt tattgcagct tataatggtt acaaataaag
     4921 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     4981 gtccaaactc atcaatgtat cttatcatgt ctggatccgc tgcattaatg aatcggccaa
     5041 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5101 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5161 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5221 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5281 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5341 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5401 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc
     5461 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5521 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5581 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5641 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     5701 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5761 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5821 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5881 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5941 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6001 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6061 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6121 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6181 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6241 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6301 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6361 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6421 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6481 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6541 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6601 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6661 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6721 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6781 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6841 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6901 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6961 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctga
//
