LOCUS       Exported                5986 bp ds-DNA     circular SYN 02-DEC-2021
DEFINITION  One of three plasmids required to create CDK1as human cells by 
            single transfection (One shot system). CDK1as cDNA and Zeocin 
            resistance cassette flanked by Sleeping Beauty transposon..
ACCESSION   .
VERSION     .
KEYWORDS    CDK1as_T2A_Zeo
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5986)
  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 5986)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 2, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5986
                     /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..2618
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    2690..2824
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2727..2746)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2781..2800
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3247..3267)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3247..3265)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3286..3302)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3286..3302)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3299..3321)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3310..3326
                     /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(3334..3364)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3379..3400
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3517..3534)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3688..4276)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3768..3787)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4447..5307)
                     /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     5070..5089
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5308..5412)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5439..5894
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5526..5545)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5736..5757
                     /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 cccatggcca agttgaccag tgccgttccg gtgctcaccg
     2281 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact
     2341 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg
     2401 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg
     2461 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg
     2521 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg
     2581 gcaactgcgt gcacttcgtg gccgaggagc aggactgatc tagagccggg acgccggctg
     2641 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     2701 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2761 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     2821 gatcccttga aatacatcca caggtacacc tccaattgac tcaaatgatg tcaattagtc
     2881 tatcagaagc ttgtggaagg ctactcgaaa tgtttgaccc aagttaaaca atttaaaggc
     2941 aatgctacca aatactaatt gagtgtatgt aaacttctga cccactggga atgtgatgaa
     3001 agaaataaaa gctgaaatga atcattctct ctactattat tctgatattt cacattctta
     3061 aaataaagtg gtgatcctaa ctgacctaag acagggaatt tttactagga ttaaatgtca
     3121 ggaattgtga aaaagtgagt ttaaatgtat ttggctaagg tgtatgtaaa cttccgactt
     3181 caactgtata gggatcctct agctagagtc gacctcgagg gggggcccgg tacccagctt
     3241 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     3301 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     3361 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     3421 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3481 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3541 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3601 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3661 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3721 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3781 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3841 cctgtccgcc tttctccctt cgggaaccgt ggcgctttct catagctcac gctgtaggta
     3901 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3961 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4021 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4081 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4141 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4201 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4261 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4321 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4381 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4441 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4501 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4561 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4621 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4681 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4741 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4801 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4861 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaacttgg ccgcagtgtt
     4921 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4981 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5041 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5101 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5161 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5221 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5281 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5341 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5401 aggggttccg cgcacatttc cccgaaaagt gccacctgac gcgccctgta gcggcgcatt
     5461 aagcgcggcg ggagtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     5521 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5581 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5641 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5701 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5761 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     5821 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5881 aacgcttaca atttccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     5941 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctg
//
