LOCUS       Exported                6211 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  One of three plasmids required to create CDK1as human cells by 
            single transfection (One shot system). CDK1as cDNA and puromycin 
            resistance cassette flanked by Sleeping Beauty transposon..
ACCESSION   .
VERSION     .
KEYWORDS    CDK1as_T2A_puromycin
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6211)
  AUTHORS   Saldivar JC, Hamperl S, Bocek MJ, Chung M, Bass TE, 
            Cisneros-Soberanis F, Samejima K, Xie L, Paulson JR, Earnshaw WC, 
            Cortez D, Meyer T, Cimprich KA
  TITLE     An intrinsic S/G2 checkpoint enforced by ATR.
  JOURNAL   Science. 2018 Aug 24;361(6404):806-810. doi: 
            10.1126/science.aap9346.
  PUBMED    30139873
REFERENCE   2  (bases 1 to 6211)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6211
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     34..56
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     48..65
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     49..65
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        75..93
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        510..889
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        890..1093
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1044..1064
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        1138..1156
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2190..2243
                     /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             2244..2843
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2244..2263)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2740..2760
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2915..3049
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2952..2971)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3006..3025
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3472..3492)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3472..3490)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3511..3527)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3511..3527)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3524..3546)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3535..3551
                     /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(3559..3589)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3604..3625
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3742..3759)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3913..4501)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3993..4012)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4672..5532)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAAKLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5295..5314
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5533..5637)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5664..6119
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5751..5770)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5961..5982
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
       61 ccagtgagcg cgcgtaatac gactcactat agggcgaatt ggagctcgga tccctataca
      121 gttgaagtcg gaagtttaca tacacttaag ttggagtcat taaaactcgt ttttcaacta
      181 ctccacaaat ttcttgttaa caaacaatag ttttggcaag tcagttagga catctacttt
      241 gtgcatgaca caagtcattt ttccaacaat tgtttacaga cagattattt cacttataat
      301 tcactgtatc acaattccag tgggtcagaa gtttacatac actaagttga ctgtgccttt
      361 aaacagcttg gaaaattcca gaaaatgatg tcatggcttt agaagcttcc cacaataagt
      421 tgggtgaatt ttggctcatt cctcctttct ataggattga ggtcagagct ttgtgatgga
      481 attcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
      541 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      601 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      661 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      721 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      781 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      841 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      901 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      961 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     1021 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     1081 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
     1141 tacgactcac tatagggaga cccaagcttg gtaccgagct cggatccact agtaacggcc
     1201 gccagtgtgc tggaattccg gaaacagcca tggacgagta cactaaaata gaaaagatcg
     1261 gagagggcac atatggggtt gtgtacaaag gtcgtcacaa agcaactggc caggttgttg
     1321 caatgaagaa aatccgattg gaaaacgaag aggaaggtgt cccaagtaca gcaatccgag
     1381 aaatatcact acttaaagag cttcagcacc ctaacatagt ttgtctccta gatgtcctca
     1441 tgcaagattc aaggttgtat cttatcggtg agtttctctc catggatcta aagaagtatt
     1501 tggactccat acccagcggc cagtatattg atacgatgct cgttaagagt tacctatacc
     1561 agatcctaca aggcattgta ttttgccact ccagaagagt gctacacaga gacctgaaac
     1621 ctcagaacct gctcatcgac agcaagggag tgataaagtt ggcagatttt gggcttgcca
     1681 gagctttcgg cattccagtt cgggtttaca cacatgaggt agtgacatta tggtacagag
     1741 ccccagaagt gctgttgggg tcagtccgat attctacgcc ggttgatgtt tggagcatag
     1801 gaaccatttt tgcggagatt gccacaaaga aacccctctt ccacggggac tctgaaattg
     1861 accagctctt caggatattc agagctttgg gaacacccaa caatgaggtg tggccagaag
     1921 tagaatcttt acaagattac aagaactcat tccccaaatg gaaagggggg agcttgtcgg
     1981 cgaacgtgaa aaatatcgat aaggatgggc tggacctact gactaaaatg ctaatctatg
     2041 atccagccaa gagaatttcc gcacgtaaag ctttgctgca cccctacttc gatgatttgg
     2101 ataagtccag ccttcctgac aatcagatta gaaactccat ggagcagaag ctgatctccg
     2161 aggaggacat ccatcacact ggcggccgcg agggccgcgg cagcctgctg acctgcggcg
     2221 acgtggagga gaaccccggc cccatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     2281 gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     2341 cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     2401 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     2461 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     2521 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     2581 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
     2641 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     2701 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     2761 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     2821 tgacccgcaa gcccggtgcc tgatctagag ccgggacgcc ggctggatga tcctccagcg
     2881 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     2941 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3001 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcc cttgaaatac
     3061 atccacaggt acacctccaa ttgactcaaa tgatgtcaat tagtctatca gaagcttgtg
     3121 gaaggctact cgaaatgttt gacccaagtt aaacaattta aaggcaatgc taccaaatac
     3181 taattgagtg tatgtaaact tctgacccac tgggaatgtg atgaaagaaa taaaagctga
     3241 aatgaatcat tctctctact attattctga tatttcacat tcttaaaata aagtggtgat
     3301 cctaactgac ctaagacagg gaatttttac taggattaaa tgtcaggaat tgtgaaaaag
     3361 tgagtttaaa tgtatttggc taaggtgtat gtaaacttcc gacttcaact gtatagggat
     3421 cctctagcta gagtcgacct cgaggggggg cccggtaccc agcttttgtt ccctttagtg
     3481 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3541 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     3601 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3661 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3721 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     3781 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     3841 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3901 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3961 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4021 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4081 cccttcggga accgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4141 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4201 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4261 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4321 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4381 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4441 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4501 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4561 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4621 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4681 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4741 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     4801 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     4861 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     4921 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     4981 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5041 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5101 cttcggtcct ccgatcgttg tcagaagtaa cttggccgca gtgttatcac tcatggttat
     5161 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5221 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5281 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5341 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5401 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5461 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5521 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5581 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5641 atttccccga aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg cggcgggagt
     5701 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     5761 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     5821 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     5881 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5941 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     6001 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     6061 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc
     6121 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     6181 ttacgccagc tggcgaaagg gggatgtgct g
//
