LOCUS       Exported                6646 bp ds-DNA     circular SYN 13-SEP-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pXK-607 pXL-BacII-CMV-PuroR(ATAACG code shift)-T2A-EGFP-sgT
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6646)
  AUTHORS   Triple Threat
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Sep 14, 2023 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6646
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     6..20
                     /label=5027-F MluI
     enhancer        98..401
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     394..413
                     /label=CMV-F NcoI
     promoter        402..605
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(605..651)
                     /label=CMV-overlap-R
     primer_bind     complement(605..651)
                     /label=CMV-overlap-R（ATAACG）
     primer_bind     627..675
                     /label=puro-overlap-F（ATAACG）
     misc_feature    639..658
                     /label=(ATAACG)sg-T
     CDS             641..1240
                     /codon_start=1
                     /label=PuroR
                     /translation="ITECKPRVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGAM"
     primer_bind     complement(1224..1255)
                     /label=puro-R(AgeI)
     CDS             1262..1315
                     /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             1316..2032
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     polyA_signal    2150..2271
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     2272..2297
                     /label=Primer 1
     promoter        2278..2526
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    2527..2546
                     /label=(ATAACG)sgRNA
     misc_RNA        2547..2622
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2684..2699)
                     /label=5027-R BamH1-Mlu1
     primer_bind     complement(2693..2711)
                     /label=5027-R SpeI
     primer_bind     complement(2697..2718)
                     /label=Primer 2
     misc_feature    2715..3024
                     /label=5' TR
     rep_origin      complement(3913..4501)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     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
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5533..5637)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5663..6118)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     6260..6276
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    6402..6636
                     /label=3' TR
ORIGIN
        1 aattcacggc ctcgacattg attattgact agagcattag ttattaatag taatcaatta
       61 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      121 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      181 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      241 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      301 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      361 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      421 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      481 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      541 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      601 gagctggttt agtgaaccgt cagatcccgc taccgccaat ataacggaat gcaagcccag
      661 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
      721 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
      781 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
      841 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
      901 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
      961 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     1021 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     1081 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
     1141 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     1201 accgcgcacc tggtgcatga cccgcaagcc cggtgccatg cgagctaccg gtggcagtgg
     1261 agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccagtgag
     1321 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     1381 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     1441 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     1501 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     1561 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     1621 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     1681 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     1741 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     1801 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     1861 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     1921 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     1981 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aatctagata
     2041 actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     2101 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     2161 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2221 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aaagcttgag
     2281 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
     2341 attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
     2401 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
     2461 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
     2521 aacaccatat aacggaatgc aagcccgttt tagagctaga aatagcaagt taaaataagg
     2581 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt tttagagcta
     2641 gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat tctgcagaca
     2701 aatggctcta ggatatctat aacaagaaaa tatatatata ataagttatc acgtaagtag
     2761 aacatgaaat aacaatataa ttatcgtatg agttaaatct taaaagtcac gtaaaagata
     2821 atcatgcgtc attttgactc acgcggtcgt tatagttcaa aatcagtgac acttaccgca
     2881 ttgacaagca cgcctcacgg gagctccaag cggcgactga gatgtcctaa atgcacagcg
     2941 acggattcgc gctatttaga aagagagagc aatatttcaa gaatgcatgc gtcaatttta
     3001 cgcagactat ctttctaggg ttaatctagc tgcatcagga tcatatcgtc gggtcttttt
     3061 tccggctcag tcatcgccca agctggcgct atctgggcat cggggaggaa gaagcccgtg
     3121 ccttttcccg cgaggttgaa gcggcatgga aagagtttgc cgaggatgac tgctgctgca
     3181 ttgacgttga gcgaaaacgc acgtttacca tgatgattcg ggaaggtgtg gccatgcacg
     3241 cctttaacgg tgaactgttc gttcaggcca cctgggatac cagttcgtcg cggcttttcc
     3301 ggacacagtt ccggatggtc agcccgaagc gcatcagcaa cccgaacaat accggcgaca
     3361 gccggaactg ccgtgccggt gtgcagatta atgacagcgg tgcggcgctg ggatattacg
     3421 tcagcgagga cgggtatcct ggctggatgc cgcagaaatg gacatggata ccccgtgagt
     3481 tacccggcgg 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 agcgtggcgc 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 aaggacagta 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 gttggccgca 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 ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     5701 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     5761 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     5821 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     5881 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     5941 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     6001 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     6061 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     6121 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     6181 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     6241 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcctcgtt cattcacgtt
     6301 tttgaacccg tggaggacgg gcagactcgc ggtgcaaatg tgttttacag cgtgatggag
     6361 cagatgaaga tgctcgacac gctgcagaac acgcagctag attaacccta gaaagataat
     6421 catattgtga cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg ttttatcggt
     6481 ctgtatatcg aggtttattt attaatttga atagatatta agttttatta tatttacact
     6541 tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa
     6601 aacaaaaact caaaatttct tctataaagt aacaaaactt ttatcg
//
