LOCUS       pNCM24_(pCMV6-AN        8060 bp DNA     circular SYN 26-MAY-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8060)
  AUTHORS   nichd_dir_cssc_sub
  TITLE     Direct Submission
  JOURNAL   Exported May 26, 2024 from SnapGene Viewer 7.2.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8060
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..25
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; 
                     arrowindicates direction of (+) strand synthesis"
     primer_bind     166..182
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        343..722
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        723..926
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate earlypromoter"
     promoter        952..970
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1014..1023
                     /note="vertebrate consensus sequence for strong 
                     initiationof translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1023..1046
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an 
                     enterokinasecleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1065..3203
                     /codon_start=1
                     /label=K709A POLH CDS
                     /label=HS-CO-eta
                     /note="FLAG-Tagged_HS-CO-eta"
                     /translation="MATGQDRVVALVDMDCFFVQVEQRQNPHLRNKPCAVVQYKSWKGG
                     GIIAVSYEARAFGVTRSMWADDAKKLCPDLLLAQVRESRGKANLTKYREASVEVMEIMS
                     RFAVIERASIDEAYVDLTSAVQERLQKLQGQPISADLLPSTYIEGLPQGPTTAEETVQK
                     EGMRKQGLFQWLDSLQIDNLTSPDLQLTVGAVIVEEMRAAIERETGFQCSAGISHNKVL
                     AKLACGLNKPNRQTLVSHGSVPQLFSQMPIRKIRSLGGKLGASVIEILGIEYMGELTQF
                     TESQLQSHFGEKNGSWLYAMCRGIEHDPVKPRQLPKTIGCSKNFPGKTALATREQVQWW
                     LLQLAQELEERLTKDRNDNDRVATQLVVSIRVQGDKRLSSLRRCCALTRYDAHKMSHDA
                     FTVIKNCNTSGIQTEWSPPLTMLFLCATKFSASAPSSSTDITSFLSSDPSSLPKVPVTS
                     SEAKTQGSGPAVTATKKATTSLESFFQKAAERQKVKEASLSSLTAPTQAPMSNSPSKPS
                     LPFQTSQSTGTEPFFKQKSLLLKQKQLNNSSVSSPQQNPWSNCKALPNSLPTEYPGCVP
                     VCEGVSKLEESSKATPAEMDLAHNSQSMHASSASKSVLEVTQKATPNPSLLAAEDQVPC
                     EKCGSLVPVWDMPEHMDYHFALELQKSFLQPHSSNPQVVSAVSHQGKRNPKSPLACTNK
                     RPRPEGMQTLESFFAPLTH"
     misc_feature    3153..3158
                     /label=Silent StuI
     misc_feature    3189..3191
                     /label=K709A
     primer_bind     complement(3268..3284)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    3295..3917
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     promoter        3946..4275
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4126..4261
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4342..5136
                     /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    5310..5431
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5480..5496)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5504..5520
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator 
                     toinhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(5528..5558)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5573..5594
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the 
                     presenceof cAMP."
     rep_origin      complement(5882..6467)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6638..7498)
                     /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(7499..7603)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7630..8060
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; 
                     arrowindicates direction of (+) strand synthesis"
ORIGIN
        1 aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag
       61 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
      121 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
      181 gtgccaagct gatctataca ttgaatcaat attggcaatt agccatatta gtcattggtt
      241 atatagcata aatcaatatt ggctattggc cattgcatac gttgtatcta tatcataata
      301 tgtacattta tattggctca tgtccaatat gaccgccatg ttgacattga ttattgacta
      361 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      421 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      481 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      541 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      601 gtccgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      661 tgaccttacg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      721 tggtgatgcg gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat
      781 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      841 actttccaaa atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac
      901 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagaatttt gtaatacgac
      961 tcactatagg gcggccggga attcgtcgac tggatccggt accgaggaga tctgccacca
     1021 tggattacaa ggatgacgac gataaggcga tcgccgatat ccatatggct acaggacagg
     1081 atagagtcgt cgcactggtg gatatggatt gtttcttcgt gcaggtggag cagcggcaga
     1141 accctcatct gcgcaacaag ccatgcgctg tggtccagta caagtcctgg aaaggcgggg
     1201 gaatcattgc agtgtcttat gaggccagag ctttcggagt caccaggagc atgtgggccg
     1261 acgatgccaa gaagctgtgc cccgacctgc tgctggccca ggtgagggag tcccgcggca
     1321 aggcaaatct gacaaaatac cgagaagcca gtgtggaggt catggaaatc atgtcacgct
     1381 ttgccgtgat cgagcgagct agtattgacg aggcctacgt ggacctgacc agcgccgtgc
     1441 aggagcggct ccagaagctc cagggacagc caatctcagc cgacctgctg cctagcacct
     1501 acattgaggg gctgcctcag ggaccaacca cagccgagga aacagtgcag aaggaaggaa
     1561 tgcgcaaaca gggcctgttc cagtggctgg acagcctcca gatcgataac ctgacatccc
     1621 ccgacctcca gctgactgtg ggagcagtga tcgtcgagga aatgagagcc gctattgaga
     1681 gggaaacagg cttccagtgc agcgccggga tttcccacaa taaggtgctg gcaaaactgg
     1741 cctgtggcct gaacaagcct aatcggcaga ctctggtgtc acacgggagc gtcccacagc
     1801 tgttttccca gatgcccatc cgaaagattc ggtctctggg cgggaaactg ggcgccagtg
     1861 tgatcgagat tctggggatc gagtacatgg gagaactgac tcagttcacc gagtctcagc
     1921 tccagagtca ctttggcgaa aagaacgggt cttggctgta tgccatgtgc agggggatcg
     1981 agcatgatcc cgtgaagcct cgccagctgc ctaaaaccat tggatgtagc aagaattttc
     2041 caggcaaaac tgcactggcc accagagagc aggtgcagtg gtggctgctc cagctggccc
     2101 aggaactgga ggaacgcctg actaaggacc gaaacgacaa tgatcgggtg gctacccagc
     2161 tggtggtcag catccgggtc cagggggata aaagactgag ctccctgcgg agatgctgtg
     2221 ccctgacaag gtacgacgct cacaagatgt cccatgatgc tttcactgtg atcaaaaact
     2281 gcaatacctc cggcatccag acagagtggt ctccccctct gaccatgctg ttcctgtgcg
     2341 ccacaaagtt tagtgcttca gcaccctcta gttcaacaga cattacttct tttctgagct
     2401 ccgatccttc tagtctgcca aaggtgcccg tcacttcaag cgaggctaaa acccagggaa
     2461 gcggccccgc tgtgacagca actaagaaag caactacctc cctggaatct ttctttcaga
     2521 aggcagccga gagacagaag gtcaaagaag ccagcctgtc ctctctgacc gctcccacac
     2581 aggcacctat gagcaactcc ccttctaaac catctctgcc cttccagacc agtcagtcaa
     2641 ccggcacaga gcccttcttt aagcagaaaa gtctgctgct gaagcagaaa cagctgaaca
     2701 atagttcagt gagctcccca cagcagaacc cctggtcaaa ttgcaaggcc ctgcctaata
     2761 gcctgccaac tgagtacccc gggtgcgtgc ctgtctgtga aggagtgagc aagctggagg
     2821 aatctagtaa agccacccct gctgagatgg acctggctca caacagccag tccatgcatg
     2881 ctagctccgc ttctaagagt gttctggaag tcacacagaa agccactcca aatcccagcc
     2941 tgctggccgc cgaggatcag gtgccatgcg aaaagtgtgg cagcctggtg cccgtgtggg
     3001 acatgccaga gcacatggat tatcatttcg ccctggaact ccagaagagc ttcctccagc
     3061 ctcactcctc taacccacag gtggtcagtg ccgtgtcaca tcagggcaag aggaacccta
     3121 aatccccact ggcttgtaca aataagagac caaggcctga aggaatgcag accctggaga
     3181 gctttttcgc accgttaact cactaaggat ccaagcttct cgagacgcgt taagcggccg
     3241 cactcgaggt ttaaacggcc ggccgcggtc atagctgttt cctgaacaga tcccgggtgg
     3301 catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc
     3361 accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat
     3421 aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt
     3481 agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact
     3541 gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga
     3601 ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc
     3661 accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct
     3721 cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgatttta
     3781 aaataactat accagcagga ggacgtccag acacagcata ggctacctgg ccatgcccaa
     3841 ccggtgggac atttgagttg cttgcttggc actgtcctct catgcgttgg gtccactcag
     3901 tagatgcctg ttgaattggg tacgcggcca gcttggctgt ggaatgtgtg tcagttaggg
     3961 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4021 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4081 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     4141 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4201 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4261 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     4321 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     4381 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     4441 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     4501 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     4561 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     4621 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     4681 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4741 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     4801 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4861 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4921 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4981 gtggccgacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5041 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     5101 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     5161 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     5221 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     5281 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     5341 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5401 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     5461 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5521 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5581 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5641 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5701 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5761 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5821 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5881 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5941 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     6001 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6061 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6121 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6181 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6241 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6301 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     6361 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtt
     6421 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6481 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6541 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6601 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6661 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6721 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6781 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6841 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6901 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6961 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     7021 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7081 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7141 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7201 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7261 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7321 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7381 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7441 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7501 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7561 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7621 tgccacctga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     7681 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     7741 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     7801 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     7861 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     7921 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     7981 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     8041 aaaaatttaa cgcgaatttt
//
