LOCUS       pNCM18_(NEDD8_OH        5690 bp DNA     circular SYN 24-MAY-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5690)
  AUTHORS   nichd_dir_cssc_sub
  TITLE     Direct Submission
  JOURNAL   Exported May 24, 2024 from SnapGene Viewer 7.2.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5690
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             926..952
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             959..1201
                     /codon_start=1
                     /label=NEDD8 CDS
                     /translation="MLIKVKTLTGKEIEIDIEPTDKVERIKERVEEKEGIPPQQQRLIY
                     SGKQMNDEKTAADYKILGGSVLHLVLALRGGGGLRQ"
     misc_feature    1181..1186
                     /label=-GG conjugated to substrates
     propeptide      1187..1201
                     /label=Cleaved to produre mature NEDD8
     polyA_signal    1287..1511
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1557..1985
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        1999..2328
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2179..2314
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2395..3189
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3363..3484
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3533..3549)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3557..3573
                     /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)."
     promoter        complement(join(3581..3587,3588..3605,3606..3611))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3626..3647
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3935..4523)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4694..5485,5486..5554))
                     /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(5555..5659)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcca ccatgtaccc atacgatgtt ccagattacg ctggtaccat
      961 gctaattaaa gtgaagacgc tgaccggaaa ggagattgag attgacattg aacctacaga
     1021 caaggtggag cgaatcaagg agcgtgtgga ggagaaagag ggaatccccc cacaacagca
     1081 gaggctcatc tacagtggca agcagatgaa tgatgagaag acagcagctg attacaagat
     1141 tttaggtggt tcagtccttc acctggtgtt ggctctgaga ggaggaggtg gtcttaggca
     1201 gtgaggatcc gaattctgca gatatccagc acagtggcgg ccgctcgagt ctagagggcc
     1261 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     1321 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     1381 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     1441 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     1501 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     1561 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1621 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1681 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1741 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1801 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1861 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     1921 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     1981 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     2041 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     2101 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     2161 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     2221 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     2281 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     2341 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     2401 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     2461 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     2521 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     2581 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     2641 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     2701 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     2761 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     2821 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     2881 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     2941 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     3001 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     3061 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     3121 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     3181 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     3241 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     3301 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     3361 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3421 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3481 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     3541 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3601 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     3661 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     3721 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     3781 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3841 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3901 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3961 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4021 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4081 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4141 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4201 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4261 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4321 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4381 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4441 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4501 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4561 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4621 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4681 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4741 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4801 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     4861 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4921 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4981 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5041 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5101 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5161 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5221 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5281 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5341 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5401 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5461 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5521 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5581 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5641 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
