LOCUS       Exported                3813 bp DNA     circular SYN 11-SEP-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3813)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Jun 4, 2021 from SnapGene Viewer 5.3.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3813
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     18..42
                     /label=pIVTR(AG)(HBB)_Fwd
     misc_feature    21..64
                     /label=HBB 5'UTR
     misc_feature    65..100
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     80..118
                     /label=CRE_GA-OL_Fwd
     primer_bind     80..100
                     /label=Puro_GA-OL_Fwd
     CDS             118..138
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large 
                     Tantigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             139..1165
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase frombacteriophage
                     P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     primer_bind     complement(1147..1185)
                     /label=CRE_GA-OL(T2A)_Rv*
     primer_bind     1165..1181
                     /label=T2A_PstI_Fwd
     primer_bind     1165..1181
                     /label=T2A-PuroR_Fwd
     CDS             1174..1227
                     /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"
     primer_bind     complement(1187..1207)
                     /label=T2A_Screen_Rv
     primer_bind     complement(1209..1227)
                     /label=T2A_Rv
     primer_bind     1222..1252
                     /label=PuroR_GA-OL_Fwd
     CDS             1237..1836
                     /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(1237..1256)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1240..1252
                     /label=PuroR_GA-OL_Fwd
     misc_feature    1272
                     /label=C>T in #1 (synonymous)
     primer_bind     1733..1753
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1815..1833)
                     /label=Puro_F2A-GA-OL_Fwd
     primer_bind     complement(1820..1839)
                     /label=PuroR-HindIII_Fwd
     primer_bind     complement(1821..1860)
                     /label=PuroR_GA-OL(HincII)_Rv
     misc_feature    1840..1860
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     1860..1886
                     /label=HBB_3'UTR_Fwd
     3'UTR           1861..1994
                     /label=HBB 3'UTR
     primer_bind     complement(1861..1879)
                     /label=HBB_3UTR_seq_Rv
     primer_bind     complement(1958..1996)
                     /label=HBB_3UTR_150T_Rv
     primer_bind     1995..2023
                     /label=pUC19_PciI-to-NcoI_Fwd
     rep_origin      complement(2058..2646)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2817..3677)
                     /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(3678..3782)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3784..3803
                     /label=Pre_T7_Fwd
     primer_bind     complement(3790..3813)
                     /label=pUC_AatII_up
     primer_bind     complement(join(3803..3813,1..17))
                     /label=pIVTR(AG)(HBB)_Rv
     primer_bind     join(3809..3813,1..41)
                     /label=T7(AG)_IVTR_Fwd
ORIGIN
        1 taatacgact cactataagg acatttgctt ctgacacaac tgtgttcact agcaacctca
       61 aacagaattc gagctcggta cccggggatc ctctagagtc aaagaattgc cgccatgccc
      121 aagaagaaga ggaaggtgtc caatttactg accgtacacc aaaatttgcc tgcattaccg
      181 gtcgatgcaa cgagtgatga ggttcgcaag aacctgatgg acatgttcag ggatcgccag
      241 gcgttttctg agcatacctg gaaaatgctt ctgtccgttt gccggtcgtg ggcggcatgg
      301 tgcaagttga ataaccggaa atggtttccc gcagaacctg aagatgttcg cgattatctt
      361 ctatatcttc aggcgcgcgg tctggcagta aaaactatcc agcaacattt gggccagcta
      421 aacatgcttc atcgtcggtc cgggctgcca cgaccaagtg acagcaatgc tgtttcactg
      481 gttatgcggc ggatccgaaa agaaaacgtt gatgccggtg aacgtgcaaa acaggctcta
      541 gcgttcgaac gcactgattt cgaccaggtt cgttcactca tggaaaatag cgatcgctgc
      601 caggatatac gtaatctggc atttctgggg attgcttata acaccctgtt acgtatagcc
      661 gaaattgcca ggatcagggt taaagatatc tcacgtactg acggtgggag aatgttaatc
      721 catattggca gaacgaaaac gctggttagc accgcaggtg tagagaaggc acttagcctg
      781 ggggtaacta aactggtcga gcgatggatt tccgtctctg gtgtagctga tgatccgaat
      841 aactacctgt tttgccgggt cagaaaaaat ggtgttgccg cgccatctgc caccagccag
      901 ctatcaactc gcgccctgga agggattttt gaagcaactc atcgattgat ttacggcgct
      961 aaggatgact ctggtcagag atacctggcc tggtctggac acagtgcccg tgtcggagcc
     1021 gcgcgagata tggcccgcgc tggagtttca ataccggaga tcatgcaagc tggtggctgg
     1081 accaatgtaa atattgtcat gaactatatc cgtaacctgg atagtgaaac aggggcaatg
     1141 gtgcgcctgc tggaagatgg cgatggcagc ggagagggca gaggaagtct tctaacatgc
     1201 ggtgacgtgg aggagaatcc cggccctagg ctcgagatga ccgagtacaa gcccacggtg
     1261 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     1321 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     1381 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     1441 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     1501 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     1561 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     1621 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     1681 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     1741 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     1801 cgcacctggt gcatgacccg caagcccggt gcctgatctg acctgcaggc atgcaagctt
     1861 gctcgctttc ttgctgtcca atttctatta aaggttcctt tgttccctaa gtccaactac
     1921 taaactgggg gatattatga agggccttga gcatctggat tctgcctaat aaaaaacatt
     1981 tattttcatt gcaaaaccat gggagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     2041 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2101 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2161 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2221 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     2281 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2341 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2401 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2461 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     2521 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     2581 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     2641 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2701 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2761 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2821 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2881 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     2941 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     3001 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3061 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3121 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3181 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3241 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3301 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3361 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     3421 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     3481 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3541 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     3601 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     3661 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     3721 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     3781 cgcacatttc cccgaaaagt gccacctgac gtc
//
