LOCUS       Exported                4983 bp DNA     circular SYN 22-JUL-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 4983)
  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..4983
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     CDS             1..302
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="HACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNRLAAHPPFA
                     SWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDAA"
     misc_feature    1..52
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     misc_feature    1..20
                     /label=RIGHT HIFI HOMOLOGY
     misc_feature    1..20
                     /label=puc19_right_homology
     primer_bind     complement(1..20)
                     /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(1..20)
                     /label=CMA135
     primer_bind     complement(53..70)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(53..69)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(62..84)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(278..297)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(372..391)
                     /label=SS225
     primer_bind     397..419
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     452..469
                     /label=SS224
     primer_bind     complement(457..475)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        543..647
                     /gene="bla"
                     /label=AmpR promoter
     CDS             648..1508
                     /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"
     primer_bind     complement(866..885)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1679..2267
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1730..1744
                     /label=SS028
     primer_bind     2168..2187
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2421..2438
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2555..2576
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2566..2583)
                     /label=DS13
     promoter        2591..2621
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2629..2645
                     /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     2634..2656
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2653..2669
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2653..2669
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2663..2683
                     /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     2663..2683
                     /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     2663..2683
                     /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    2663..2682
                     /label=LEFT HIFI HOMOLOGY
     misc_feature    2663..2682
                     /label=left hifi homology
     CDS             2665..2682
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITP"
     misc_feature    2682
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     misc_feature    2683..3432
                     /label=750_bp_before_endCodon
     primer_bind     3148..3167
                     /label=DS56
     primer_bind     complement(3198..3217)
                     /label=DS36
     misc_feature    3433..3452
                     /label=283_bbs1_homology
     primer_bind     3433..3451
                     /label=AHPG226
                     /note="Oligo for CTAP tag with 20 base homology for PLD6 
                     3`end. For HIFI."
     primer_bind     3433..3451
                     /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    3433
                     /label=Clone 2 Deletion Mutation
                     /note="This base pair is deleted in clone 2"
     primer_bind     3435..3451
                     /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     3435..3450
                     /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             3436..3573
                     /codon_start=1
                     /label=3FHA_Ctag
                     /translation="SGGGSGDYKDHDGDYKDHDIDYKDDDDKAGASSAYPYDVPDYAAG
                     S"
     primer_bind     3436..3573
                     /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(3436..3453)
                     /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     3436..3453
                     /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    3436..3452
                     /label=3FHA_Ctag_HA
     primer_bind     3436..3451
                     /label=HAL-Tia1l-CTAP-F1
     primer_bind     3436..3451
                     /label=HAL-Tia1l-CTAP-F2
     primer_bind     3436..3451
                     /label=HAL-Tai1l-CTAP-F3
     primer_bind     3436..3450
                     /label=AHPG289
                     /note="Primer to amplify 3FHA_Ctag. Includes homology for 
                     (AHPG92) gB_MAEL_HA1C. Fw. Tm = 60.1C."
     primer_bind     complement(3436..3450)
                     /label=AHPG287
                     /note="Amplify V5_spacer_N-tag. Reverse oligo with homology
                     for MOV10L1 for HIFI."
     primer_bind     complement(3437..3452)
                     /label=AHPG294
                     /note="Tm = 64.2 C. To amplify gB_MAEL_HA1C. Rw."
     primer_bind     complement(3438..3452)
                     /label=AHPG91
                     /note="To amplify gBlocks for CTAP HA1 reverse."
     CDS             3454..3519
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     3454..3472
                     /label=AHPG118
                     /note="For genotyping of tagged clones - in Flag - forward"
     primer_bind     complement(3454..3472)
                     /label=AHPG119
                     /note="For genotyping of tagged clones - in Flag - reverse"
     primer_bind     3455..3472
                     /label=AHPG70
                     /note="To sequence NTAP and CTAP new pFETCh vectors. Is in 
                     the beginning of the 3xFLAG tag. Forward."
     primer_bind     complement(3455..3472)
                     /label=AHPG71
                     /note="To sequence NTAP and CTAP new pFETCh vectors. Is in 
                     the beginning of the 3xFLAG tag. Reverse."
     primer_bind     complement(3475..3497)
                     /label=SS036
     primer_bind     3498..3519
                     /label=SS035
     primer_bind     complement(3504..3546)
                     /label=SS116
     CDS             3538..3564
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3555..3613)
                     /label=AHPG290
                     /note="To amplify 3FHA_Ctag including half of P2A peptide. 
                     For cloning with PurR into pFETCh. Rw. Tm = 61.2"
     CDS             3574..3630
                     /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     3596..3648
                     /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     3626..3644
                     /label=AE9
                     /note="Tm = 58.5˚Used to sequence"
     primer_bind     complement(3626..3644)
                     /label=AE10
     CDS             3631..4230
                     /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(3766..3784)
                     /label=SS186
     primer_bind     4128..4144
                     /label=DS7
     misc_feature    4179..4180
                     /label=Clone 1 Insertion Mutation
                     /note="in clone 1 there is a G inserted in between these 
                     two base pairs "
     misc_feature    4179..4180
                     
                     /note="Clone 2 has a G inserted between these two base 
                     pairs "
     misc_feature    4179..4180
                     
                     /note="Clone 3 has a G inserted in ebtween these two base 
                     pairs "
     misc_feature    4197
                     /label=Clone 1 Deletion Mutation
                     /note="This base pair is deleted in Clone 1"
     misc_feature    4197
                     
                     /note="Clone 2 has this base pair deleted "
     misc_feature    4197
                     
                     /note="	Clone 3 has this base pair deleted "
     primer_bind     4204..4218
                     /label=DS4
     misc_feature    4207..4229
                     
                     /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!"
     misc_feature    4214..4233
                     /label=283_bbs1 homology
     primer_bind     complement(4215..4230)
                     /label=CMA19
                     /note="63.4"
     primer_bind     complement(4216..4234)
                     /label=CMA135
     primer_bind     complement(4216..4230)
                     /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(4218..4230)
                     /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."
     misc_feature    4234..4983
                     /label=750nt after Nbr endCodon
     primer_bind     complement(4388..4407)
                     /label=DS57
ORIGIN
        1 tgcatgcctg caggtcgact ctagaggatc cccgggtacc gagctcgaat tcactggccg
       61 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
      121 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
      181 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
      241 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat
      301 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
      361 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
      421 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
      481 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
      541 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
      601 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
      661 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
      721 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
      781 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
      841 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
      901 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
      961 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     1021 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     1081 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     1141 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     1201 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     1261 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     1321 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     1381 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     1441 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     1501 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     1561 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     1621 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     1681 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     1741 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     1801 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     1861 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     1921 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     1981 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     2041 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     2101 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     2161 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     2221 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     2281 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     2341 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     2401 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     2461 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     2521 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     2581 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     2641 aacaatttca cacaggaaac agctatgacc atgattacgc cagtagatgt gatatgtacc
     2701 tgaccatgga cctgcccgac gagtgcctta tcattgtgaa caaggctgat gagttcgatc
     2761 gtatgttata ccatctgcag caggagtgcg tcatatacct ggattctgaa tggatgcaga
     2821 gcgtgtgcgg agacaatcag ctgtgcgtgc tacagatcgc caccggccac aatgtctacc
     2881 tgatcgattg cctggcgcgg gagagcttac gctcggaaca ctggcgcttg ctgggcgcga
     2941 atatctttaa taacgtgaac atccgtaagg tggggttctc tatggtcagt gatctcagtg
     3001 tattgcagcg gtcactgccc ctgcaactgc gccttcaaat gcctcaccac tacttggacc
     3061 ttcgcaatct ctggttggag ttaaaaaagc agcgcttcgg cgttgaactt cccttcggca
     3121 atgttaaccg ggcaggagac gcccttacgg atctctcatt ggcatgcctt ggcaagaaac
     3181 tgaacaaatc aaaccagtgc tcaaactggg ccaatagacc gctgcgtcgc gagcagattc
     3241 tttatgcagg tgagtcaggg ataggccttt tgattgaatg tagcaagact aattttctac
     3301 ttaacagcca tcgatgcgcg gtgtttgatg ctgatctaca acacgctcat tgaacgcgtg
     3361 tccttcatac aagcggttat cgaaaagagc atcgccagca ataactttct caggcggggt
     3421 gcccatgtta agaccagcgg aggaggttcc ggtgactaca aggaccacga cggcgattat
     3481 aaggatcacg acatcgacta caaagacgac gatgacaagg ctggcgccag cagcgcctac
     3541 ccatacgacg taccagatta cgctgctgga tctgccacga acttctctct gttaaagcaa
     3601 gcaggagacg tggaagaaaa ccccggtcct atgaccgagt acaagcccac ggtgcgcctc
     3661 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     3721 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     3781 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     3841 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     3901 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     3961 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     4021 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     4081 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     4141 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagt gcccgaagga ccgcgcgacc
     4201 tggtgcatga cccgcaagcc cggtgcctga tatttgagga gctggcctct gttataccca
     4261 tgctcctttt gatggatagc gattatcacg aagacgacaa aggcgttcgc ccaacattta
     4321 ttaagtgcaa atgttatatg tagttgcaaa gagtatttaa catatattta gtgactagcc
     4381 aggcagctgg gttagtgccg agttaggcag tttgtacaca tgttacaaat cagaaggaaa
     4441 ctatgtgact gcgagcagga tatgaaagaa tgaattttat tacgttatga tttcaagctc
     4501 actgtatctc catactttaa atacttgcat tattccaatt cgtaaattcg cgtattattt
     4561 gaattcacgc atgatcaaaa ctgaataaaa gcaatgaata ggattgaaca attgtcggga
     4621 gctaaaatag ttaaaataac attggcatcg gaataatcac attacaacgt tgtgcgatta
     4681 taccagcggt gtggcgtact tgctattcca gtcccgctcg aaaacattcc gcagatcgct
     4741 tcttaaggta ttagtggtct cggtctcgaa ggtttcgctt agcaccagtc caatgccagc
     4801 ggtgtccgtg aagtagtctc cactccagtt agaagttccg atgtaagcca ctcgatctgt
     4861 aaccatgtac ttgttgtggt tgacacggcc aaagggaatc ttctcctggc tcgaatcagt
     4921 cggcactatg aatcgacgaa tttgtatatc aattttgtct tctttggatg ccaagtcctg
     4981 cag
//
