LOCUS       Exported                4573 bp DNA     circular SYN 30-SEP-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 4573)
  AUTHORS   ASTRID HAASE
  TITLE     Direct Submission
  JOURNAL   Exported May 20, 2022 from SnapGene Viewer 6.0.5
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4573
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     protein_bind    505..526
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        541..571
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    579..595
                     /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)."
     primer_bind     603..619
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     613..632
                     /label=CMA126
                     /note="Has homology to HINDIII cut site in pUC19 vector, 
                     homology arm also includes BbSI cute site (sequence: 
                     GAAGAC)Tm = 60.1"
     primer_bind     613..632
                     /label=CMA131
                     /note="Tm = 63.3Used in conjunction with CMA130used to 
                     insert C-term tag (P2A) into PuC19. Has homology to 
                     upstream HINDIII cut site in pUC19 vector, homology arm 
                     also includes BbSI cute site (sequence: GAAGAC)"
     primer_bind     613..632
                     /label=CMA134
                     /note="Tm = 63.3     End of primer has  homology to the 
                     lacZa sequence in pUC19, contains BbSI recognition site and
                      add 9 bps (AGAAACACC) because BbSI is a distance cutter 
                     (very similar to  CMA131 - just has added sequence to 
                     account for BbSI being a distance cutter)"
     CDS             615..650
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACR"
     primer_bind     631..670
                     /label=MQC82
     misc_feature    632..650
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     CDS             651..1139
                     /codon_start=1
                     /label=genome
                     /translation="RRCLCRR*GVVVLLGHHLPGAICSAPGRPTPRDRHRCTWTTSWPW
                     KLVAPSPQDPPATTRYPQC*EAPGWVATEDLESRQQLPLGKVSRLPLSRITLMGSFYSF
                     NRQKKMMRIGSPSSWAESPGSYRSASDSHFSSSDSHYSSPHYSGRPRGRGLRSRPSYLR
                     "
     primer_bind     complement(1120..1159)
                     /label=MQC83
     misc_feature    1124..1139
                     /label=STOP
     primer_bind     1139..1158
                     /label=AHPG226
                     /note="Oligo for CTAP tag with 20 base homology for PLD6 
                     3`end. For HIFI."
     primer_bind     1140..1158
                     /label=CMA134
                     /note="Tm = 63.3     End of primer has  homology to the 
                     lacZa sequence in pUC19, contains BbSI recognition site and
                      add 9 bps (AGAAACACC) because BbSI is a distance cutter 
                     (very similar to  CMA131 - just has added sequence to 
                     account for BbSI being a distance cutter)"
     misc_feature    1140
                     /label=Clone 2 Deletion Mutation
                     /note="This base pair is deleted in clone 2"
     primer_bind     1142..1158
                     /label=CMA131
                     /note="Tm = 63.3Used in conjunction with CMA130used to 
                     insert C-term tag (P2A) into PuC19. Has homology to 
                     upstream HINDIII cut site in pUC19 vector, homology arm 
                     also includes BbSI cute site (sequence: GAAGAC)"
     primer_bind     1142..1157
                     /label=CMA126
                     /note="Has homology to HINDIII cut site in pUC19 vector, 
                     homology arm also includes BbSI cute site (sequence: 
                     GAAGAC)Tm = 60.1"
     CDS             1143..1280
                     /codon_start=1
                     /label=3FHA_Ctag
                     /translation="SGGGSGDYKDHDGDYKDHDIDYKDDDDKAGASSAYPYDVPDYAAG
                     S"
     primer_bind     1143..1280
                     /label=AHPG270
                     /note="gBlock cassete to be used for tagging. Produces 5 
                     kDa tag. Preferably for C-terminus. Add TGA in PCR."
     primer_bind     complement(1143..1160)
                     /label=AHPG277
                     /note="Crick strand of the oligo to make a tag. See order 
                     in the name. Can be used for HIFI and amplified once 
                     annealed with Watson strand."
     primer_bind     1143..1160
                     /label=AHPG278
                     /note="Watson strand of the oligo to make a tag. See order 
                     in the name. Can be used for HIFI and amplified once 
                     annealed with Crick strand."
     misc_feature    1143..1159
                     /label=3FHA_Ctag_HA
     primer_bind     1143..1158
                     /label=HAL-Tia1l-CTAP-F1
     primer_bind     1143..1158
                     /label=HAL-Tia1l-CTAP-F2
     primer_bind     1143..1158
                     /label=HAL-Tai1l-CTAP-F3
     primer_bind     1143..1157
                     /label=AHPG289
                     /note="Primer to amplify 3FHA_Ctag. Includes homology for 
                     (AHPG92) gB_MAEL_HA1C. Fw. Tm = 60.1C."
     primer_bind     complement(1143..1157)
                     /label=AHPG287
                     /note="Amplify V5_spacer_N-tag. Reverse oligo with homology
                     for MOV10L1 for HIFI."
     primer_bind     complement(1144..1159)
                     /label=AHPG294
                     /note="Tm = 64.2 C. To amplify gB_MAEL_HA1C. Rw."
     primer_bind     complement(1145..1159)
                     /label=AHPG91
                     /note="To amplify gBlocks for CTAP HA1 reverse."
     CDS             1161..1226
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     1161..1179
                     /label=AHPG118
                     /note="For genotyping of tagged clones - in Flag - forward"
     primer_bind     complement(1161..1179)
                     /label=AHPG119
                     /note="For genotyping of tagged clones - in Flag - reverse"
     primer_bind     1162..1179
                     /label=AHPG70
                     /note="To sequence NTAP and CTAP new pFETCh vectors. Is in 
                     the beginning of the 3xFLAG tag. Forward."
     primer_bind     complement(1162..1179)
                     /label=AHPG71
                     /note="To sequence NTAP and CTAP new pFETCh vectors. Is in 
                     the beginning of the 3xFLAG tag. Reverse."
     primer_bind     complement(1182..1204)
                     /label=SS036
     primer_bind     1205..1226
                     /label=SS035
     primer_bind     complement(1211..1253)
                     /label=SS116
     CDS             1245..1271
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1262..1320)
                     /label=AHPG290
                     /note="To amplify 3FHA_Ctag including half of P2A peptide. 
                     For cloning with PurR into pFETCh. Rw. Tm = 61.2"
     CDS             1281..1337
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     1303..1355
                     /label=AHPG291
                     /note="To amplify PurR from pQCXP CMV/TO DEST for cloning 
                     into pFETCh. Has half of P2A. Tm = 61.5C. Fw."
     primer_bind     1333..1351
                     /label=AE9
                     /note="Tm = 58.5˚Used to sequence"
     primer_bind     complement(1333..1351)
                     /label=AE10
     CDS             1338..1937
                     /codon_start=1
                     /gene="pac from Streptomyces"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
     primer_bind     complement(1473..1491)
                     /label=SS186
     misc_feature    1886..1887
                     /label=Clone 1 Insertion Mutation
                     /note="in clone 1 there is a G inserted in between these 
                     two base pairs "
     misc_feature    1886..1887
                     
                     /note="Clone 2 has a G inserted between these two base 
                     pairs "
     misc_feature    1886..1887
                     
                     /note="Clone 3 has a G inserted in ebtween these two base 
                     pairs "
     misc_feature    1904
                     /label=Clone 1 Deletion Mutation
                     /note="This base pair is deleted in Clone 1"
     misc_feature    1904
                     
                     /note="Clone 2 has this base pair deleted "
     misc_feature    1904
                     
                     /note="	Clone 3 has this base pair deleted "
     misc_feature    1914..1936
                     
                     /note="It is necessary for the PuroR to have a 3'UTR. It 
                     can either be the endogenous 3'UTR of whatever gene you are
                     taggin C-terminally, or you can add an SV40 Poly A 
                     sequence. It just has to have one or the other, it cannot 
                     be left unpoly addenylated!"
     primer_bind     1920..1959
                     /label=MQC84
     primer_bind     complement(1922..1937)
                     /label=CMA19
                     /note="63.4"
     primer_bind     complement(1923..1939)
                     /label=CMA135
     primer_bind     complement(1923..1937)
                     /label=CMA130
                     /note="Tm = 63.1     used to insert C-term tag (P2A) into 
                     PuC19. Has homology to downstream HINDIII cut site in pUC19
                      vector, homology arm also includes BbSI cute site 
                     (sequence: GAAGAC)"
     primer_bind     complement(1925..1937)
                     /label=AHPG292
                     /note="Oligo to amplify PuroR gene from pQCXP CMV/TO Dest 
                     for cloning into pFETCh MAEL donor. Tm = 62.3C. Has HA for 
                     HA2C of MAEL. Rw."
     primer_bind     1966..1987
                     /label=MQC110
     misc_feature    1968..1987
                     /label=sgRNA
     primer_bind     complement(1969..1987)
                     /label=MQC111
     misc_feature    2002..2021
                     /label=sgRNA reverse
     primer_bind     2002..2021
                     /label=MQC113
     primer_bind     complement(2002..2021)
                     /label=MQC112
     misc_feature    2042..2061
                     /label=sgRNA reverse
     primer_bind     2042..2060
                     /label=MQC115
     primer_bind     complement(2042..2060)
                     /label=MQC114
     primer_bind     complement(2528..2567)
                     /label=MQC85
     CDS             2548..2825
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="DPRVPSSNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEA
                     RTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDAA"
     misc_feature    2548..2575
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2576..2592)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3066..3170
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3171..4031
                     /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"
     rep_origin      join(4202..4573,1..217)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
       61 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      241 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
      301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
      361 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
      421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      481 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
      541 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      601 cacaggaaac agctatgacc atgattacgc caagcttgca tgcctgcagg agaagatgtt
      661 tgtgtcgtcg gtaaggggtc gtggttttgc tcggccacca ccttccaggg gcgatttgtt
      721 ccgctcccgg ccgcccaaca cctcgcgacc gccatcgctg cacgtggacg acttcctggc
      781 cttggaaact tgtggcgccc agcccacagg acccaccggc tacaacaaga taccctcaat
      841 gctgagaggc tcccgggtgg gtcgcaaccg aggatctcga atctcggcag cagctgcctt
      901 taggtaaagt gtcacgatta ccacttagca ggataacgct aatgggctct ttttactctt
      961 ttaacaggca aaagaagatg atgcgcattg gttcgccctc cagctgggca gagtcccccg
     1021 gatcgtatcg gtctgcctcg gactcccact tcagtagcag cgactcgcac tattcgtcgc
     1081 cccactacag tggacggccg cgtggacgag gcctgcgttc caggccatcc tacctgagaa
     1141 ccagcggagg aggttccggt gactacaagg accacgacgg cgattataag gatcacgaca
     1201 tcgactacaa agacgacgat gacaaggctg gcgccagcag cgcctaccca tacgacgtac
     1261 cagattacgc tgctggatct gccacgaact tctctctgtt aaagcaagca ggagacgtgg
     1321 aagaaaaccc cggtcctatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     1381 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     1441 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     1501 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     1561 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     1621 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     1681 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     1741 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     1801 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     1861 gcttcaccgt caccgccgac gtcgagtgcc cgaaggaccg cgcgacctgg tgcatgaccc
     1921 gcaagcccgg tgcctgatag aggacattgc agcccaaccc gcttctcact gacacttccc
     1981 gtaaactcgg caaacgaacc agaactagat cctaagatgc ccgctgggca gctgaattcc
     2041 aatacttaat agtgtgtgtt gctttcgtat tagttcgctt tacttcccac tttgtatgta
     2101 agccaaaagt tagttttccg taattacact gtgcaaacgg acgtcgtgcg actccatggg
     2161 caactcctcg ttgctgacta tctgctcgtt gagcgacagt gccatcagcg agatcttctt
     2221 gtgcggatac ttctccgcgt gctgcttgag gaacttgtcg tagtagccca tgccatggcc
     2281 catccgagct ccgcagcggg tgaaggccac accgggcaca atgaagagat cgatgccgtg
     2341 ccctgtggtg atcgagtgtg tgtgtgtggg tttagaagga gctctgatgt atgcctagcc
     2401 actcaccgtt ggtcatggca tcctcgcgtg cctccttgaa gtccggctgc tttatgttcc
     2461 acttggtcag aggcaggctc tcgtactcct ccatgccgcg cagccgcacc atcttcatcc
     2521 tgctgccctc gtaggtgggc acaaagaagg atccccgggt accgagctcg aattcactgg
     2581 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     2641 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     2701 cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
     2761 atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
     2821 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     2881 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     2941 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     3001 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     3061 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3121 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3181 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3241 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3301 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     3361 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     3421 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     3481 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3541 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     3601 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3661 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     3721 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3781 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     3841 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     3901 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     3961 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4021 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4081 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4141 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4201 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4261 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4321 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     4381 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4441 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4501 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4561 cctacaccga act
//
