LOCUS       Exported                3556 bp DNA     circular SYN 24-OCT-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 3556)
  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..3556
                     /organism="recombinant plasmid"
                     /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
     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     787..806
                     /label=CRE_seq(Ct)_Fwd
     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     1207..1247
                     /label=BleoR_GA-OL(T2A)_Fwd
     primer_bind     complement(1209..1227)
                     /label=T2A_Rv
     primer_bind     1219..1247
                     /label=FE2Ableo
     CDS             1228..1599
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR (codons adjusted to lower GCs)
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     primer_bind     complement(1580..1599)
                     /label=RbleoT2A
     primer_bind     complement(1582..1616)
                     /label=BleoR_GA-OL(attB2)_Rv
     primer_bind     complement(1583..1623)
                     /label=BleoR_GA-OL(HBB)_Rv
     protein_bind    complement(1601)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     1603..1629
                     /label=HBB_3'UTR_Fwd
     misc_feature    1603
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     3'UTR           1604..1737
                     /label=HBB 3'UTR
     primer_bind     complement(1604..1622)
                     /label=HBB_3UTR_seq_Rv
     primer_bind     complement(1701..1739)
                     /label=HBB_3UTR_150T_Rv
     primer_bind     1738..1766
                     /label=pUC19_PciI-to-NcoI_Fwd
     rep_origin      complement(1801..2389)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2560..3420)
                     /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(3421..3525)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3527..3546
                     /label=Pre_T7_Fwd
     primer_bind     complement(3533..3556)
                     /label=pUC_AatII_up
     primer_bind     complement(join(3546..3556,1..17))
                     /label=pIVTR(AG)(HBB)_Rv
     primer_bind     join(3552..3556,1..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 cggccctatg gccaagttga ccagtgccgt tccggtgctc
     1261 accgcgagag acgtcgccgg agcggtcgag ttctggaccg acagactcgg gttctccaga
     1321 gacttcgtgg aggacgactt cgccggtgtg gtcagagacg acgtgaccct gttcatcagc
     1381 gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt gagaggcctg
     1441 gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttcagaga cgcctccgga
     1501 ccggccatga ccgagatcgg cgagcagccg tggggaagag agttcgccct gagagacccg
     1561 gccggcaact gcgtgcactt cgtggccgag gagcaggact aatgctcgct ttcttgctgt
     1621 ccaatttcta ttaaaggttc ctttgttccc taagtccaac tactaaactg ggggatatta
     1681 tgaagggcct tgagcatctg gattctgcct aataaaaaac atttattttc attgcaaaac
     1741 catgggagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     1801 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     1861 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     1921 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     1981 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     2041 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     2101 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     2161 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     2221 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     2281 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     2341 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     2401 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     2461 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     2521 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     2581 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     2641 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     2701 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     2761 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     2821 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     2881 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     2941 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     3001 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     3061 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     3121 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     3181 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     3241 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     3301 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     3361 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     3421 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     3481 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     3541 agtgccacct gacgtc
//
