LOCUS       #49.unipolyA.Cdt        9640 bp DNA     circular SYN 26-MAY-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 9640)
  AUTHORS   Ferdinando Pucci
  TITLE     Direct Submission
  JOURNAL   Exported Sep 9, 2024 from SnapGene Viewer 7.2.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9640
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     attenuator      80..285
                     /label=40polyA oriR
     CDS             286..446
                     /label=C19
     misc_feature    447..905
                     /label=ageF1 origin
     misc_feature    906..2904
                     /label=C19
     primer_bind     3055..3075
                     /label=p1074 F primer 1 3050-3550
     attenuator      3122..3794
                     /label=V IE-I prom
     primer_bind     3412..3432
                     /label=p1074 F primer 2 3450-3950
     primer_bind     3726..3746
                     /label=p1074 F primer 3 3850-4350
     enhancer        3796..3977
                     /label=5
     attenuator      3978..4128
                     /label=S SL123
     misc_feature    4129..4483
                     /label=4 mgag
     primer_bind     4242..4262
                     /label=p1074 F primer 4 4250-4750
     misc_feature    4639..4879
                     /label=RRE
     primer_bind     4646..4666
                     /label=p1074 F primer 5 4250-4750
     primer_bind     4964..4984
                     /label=p1074 F primer 6 5050-5550
     primer_bind     5339..5359
                     /label=p1074 F primer 7 5450-5950
     misc_feature    complement(5502..5757)
                     /label=40 polyA unidirectional
     CDS             complement(5823..6095)
                     /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             complement(6102..6794)
                     /codon_start=1
                     /label=mScarlett
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     regulatory      6794..6803
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6917..7025)
                     /label=mCMV
     primer_bind     7033..7053
                     /label=p1074 F primer 11 7050-7550
     enhancer        7059..7573
                     /label=PGK
     primer_bind     7544..7564
                     /label=p1074 F primer 12 7450-7950
     regulatory      7587..7596
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7593..7595,7596..7598,7599..8309)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             8316..8645
                     /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     8649..8668
                     /label=p1074 F primer 14 8250-8750
     misc_feature    8650..9161
                     /label=WPRE*
                     /note="6bp downstream SacII-site removed by linker insert 
                     (partial homology retained though)"
     primer_bind     9012..9032
                     /label=p1074 F primer 15 8650-9150
     primer_bind     9242..9262
                     /label=p1074 F primer 16 9050-9550
     enhancer        9286..9520
                     /label=3RU5
ORIGIN
        1 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
       61 caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc cagttccgcc
      121 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
      181 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcgtc
      241 gagacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
      301 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
      361 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
      421 tgcgcagcct gaatggcgaa tggcgcgacg cgccctgtag cggcgcatta agcgcggcgg
      481 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
      541 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
      601 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
      661 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
      721 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
      781 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
      841 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
      901 tttcccaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
      961 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     1021 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     1081 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     1141 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     1201 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     1261 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     1321 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     1381 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     1441 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     1501 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     1561 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     1621 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     1681 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     1741 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     1801 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     1861 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     1921 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     1981 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2041 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2101 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2161 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     2221 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2281 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2341 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2401 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2461 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     2521 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     2581 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     2641 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     2701 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     2761 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     2821 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     2881 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     2941 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     3001 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     3061 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc
     3121 ttggccattg catacgttgt atccatatca taatatgtac atttatattg gctcatgtcc
     3181 aacattaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg
     3241 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     3301 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     3361 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     3421 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     3481 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     3541 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     3601 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     3661 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     3721 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     3781 tcgtttagtg aaccggggtc tctctggtta gaccagatct gagcctggga gctctctggc
     3841 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     3901 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     3961 tggaaaatct ctagcagtgg cgcccgaaca gggacctgaa agcgaaaggg aaaccagagc
     4021 tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac
     4081 tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga
     4141 gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag
     4201 ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat
     4261 tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc
     4321 tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa
     4381 ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga
     4441 tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga
     4501 cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta
     4561 aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa
     4621 aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact
     4681 atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg
     4741 cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca
     4801 gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat
     4861 caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct
     4921 tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg
     4981 gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg
     5041 caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg
     5101 tggaattggt ttaacataac aaattggctg tggtatataa aattattcat aatgatagta
     5161 ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg
     5221 cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg
     5281 cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg
     5341 aacggatctc gacggtatcg gttaactttt aaaagaaaag gggggattgg ggggtacagt
     5401 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     5461 caaattacaa aaattcaaaa ttttatcgat cacgagacta gcctcgagag atctgatcat
     5521 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     5581 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     5641 taatggttac aaataaggca atagcatcac aaatttcaca aataaggcat ttttttcact
     5701 gcattctagt tttggtttgt ccaaactcat caatgtatct tatcatgtct ggatctcaaa
     5761 tccctcggaa gctgcgcctg tcatcgaatt cctgcagccc ggtgcatgac taagctagct
     5821 tatattgtat cttgatcttg agcggatgta aggtgtggag gttggcctgc tgcaggtgta
     5881 gactttttta ttttctggcc agggctgggg caagcgggaa tgtcgggagc ttcaggggta
     5941 gagggtgaag atacttcatc tacgctgagg cgcaatctcc gccgtgcggg aggtcttgct
     6001 tggtctcgtc caggtgctgc tggtggtcga gccctttttc tgctgcctga ggtggctgaa
     6061 gcaggagctc tcaaagcagg tctggcagga gatggttcta ccttatacaa ttcatccatc
     6121 cctccggtac tgtgcctgcc ctcactccgc tcgtactgct ctaccacagt gtagtcttca
     6181 ttgtgtgagg taatatccaa tttccgatca acgttgtaag caccaggcat ctggactggc
     6241 ttcttagcct tgtaggttgt tttaaaatca gccaggtatc gtcccccatc cttcaggcgc
     6301 agggccatct taatgtcgcc tttcaagaca ccgtcttcag gatacaggcg ttctgtactg
     6361 gcttcccacc ccatggtttt cttctgcata acaggcccat caggtgggaa gtttgtccct
     6421 cgaagcttga ccttgtaaat caaggtcccg tcctccaaag atgtgtcctg tgtgacggtc
     6481 acggccccgc catcctcaaa attcattacc cgttcccatt tgaaaccctc tgggaaggac
     6541 tgtttgtaat aatcaggtat gtctgcagga tgtttggtga atgctctact cccgtacata
     6601 aattggggag agagtatgtc ccatgagaag ggaagaggtc cgcctttggt gactttcagc
     6661 ttggctgtct gtgtaccttc ataaggtcta ccttcaccct ccccttcaat ttcgaactcg
     6721 tgaccgttca tagagccttc catatgcacc ttaaacctca taaactcctt tatcacagcc
     6781 tcccccttgg ataccatggt ggcagcgctc gacttccagc tcggtccgct ttgcggactg
     6841 atggggctgc gctgcgctgc gctccagcgc cccccctgcc cgccggagct ggccgcggcc
     6901 cgaattccgc ggaggctgga tcggtcccgg tgtcttctat ggaggtcaaa acagcgtgga
     6961 tggcgtctcc aggcgatctg acggttcact aaacgagctc tgcttatata ggcctcccac
     7021 cgtacacgcc taccctcgag aagcttgata tcgaattcca cggggttggg gttgcgcctt
     7081 ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa
     7141 cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg
     7201 atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg
     7261 gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact
     7321 agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct
     7381 gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg
     7441 cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca
     7501 ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac
     7561 ctctctcccc agggggatcc accggtgcca ccatggtgtc aaagggggag gagttgttta
     7621 ctggtgttgt acccatactc gttgaactgg atggcgacgt aaatggacat aagttcagcg
     7681 tttccggtga gggggaagga gacgccactt atggcaaact gaccttgaag ttcatatgca
     7741 caacaggaaa acttcctgtt ccctggccaa ctttggtgac aactttgacc tatggcgtac
     7801 agtgcttttc tcgctatccc gatcatatga agcagcatga ctttttcaaa agcgccatgc
     7861 ctgaaggata cgtgcaagag agaaccattt tctttaagga tgatggaaac tataagaccc
     7921 gggctgaagt caaatttgag ggtgatacat tggtaaaccg gatagaattg aaggggatag
     7981 atttcaaaga agatggaaat atactcggac acaaactgga gtacaattac aacagtcaca
     8041 atgtgtacat tatggctgac aaacagaaga atggtatcaa ggttaacttt aaaattaggc
     8101 ataacataga ggacggttca gtgcagctgg ctgaccacta ccaacaaaac acccctattg
     8161 gtgacggtcc tgttctcttg ccagataacc attacttgtc cactcaatcc gccctgagca
     8221 aagaccccaa tgaaaagcgg gaccacatgg tgcttttgga gttcgttaca gctgctggca
     8281 tcactctcgg catggacgaa ctgtacaagg tggaaatgaa tccaagtatg aaacagaagc
     8341 aagaagaaat taaggaaaat attaaaaact ccagtgttcc tagacgcact ctcaagatga
     8401 ttcagcccag cgcaagcggt tcactggtag gccgggagaa cgagttgtct gcaggcttgt
     8461 ccaagagaaa gcataggaac gatcatctga ctagcacaac aagttcccca ggagtaatag
     8521 taccagagag cagcgaaaac aagaatttgg gcggcgtcac tcaggagtcc ttcgacctca
     8581 tgatcaagga gaacccatca agccaatact ggaaagaggt cgcagaaaaa cgaagaaaag
     8641 ccctgtgagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     8701 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     8761 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     8821 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     8881 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     8941 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     9001 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct
     9061 ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     9121 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctagagc
     9181 ctcttccgcg tcttcgcctt cccgggtcga gctcggtacc tttaagacca atgacttaca
     9241 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
     9301 actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga
     9361 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     9421 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     9481 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca tgtcatctta
     9541 ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta
     9601 ttgcagctta taatggttac aaataaagca atagcatcac
//
