LOCUS       Exported                7662 bp ds-DNA     circular SYN 13-NOV-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pNLF1NW-ccdB
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 7662)
  AUTHORS   Design of Biological Systems
  TITLE     Direct Submission
  JOURNAL   Exported 13 Nov 2019 from SnapGene 2.6.2
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7662
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     regulatory      1..742
                     /note="CMV immediate early enhancer promoter"
     intron          857..989
                     /note="chimeric"
     regulatory      1033..1052
                     /note="T7 promoter"
     gene            1065..1577
                     /gene="NLuc"
                     /note="NLuc"
     CDS             1065..1577
                     /codon_start=1
                     /transl_table=11
                     /gene="NLuc"
                     /product="NanoLuc luciferase"
                     /note="NLuc"
                     /note="N-terminus"
                     /protein_id="AHH41358.1"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     misc_feature    1578..1589
                     /note="N-terminal NanoLuc linker"
     misc_feature    1590..1659
                     /note="multiple cloning region"
     misc_feature    1590..1597
                     /note="SgfI site"
     misc_feature    1666
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     protein_bind    1674..1798
                     /gene="mutant version of attR"
                     /bound_moiety="LR Clonase(TM)"
                     /note="attR1"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1823..1853
                     /note="lac UV5 promoter"
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1907..2566
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /note="CmR"
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2908..3213
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /note="ccdB"
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3254..3378)
                     /gene="mutant version of attR"
                     /bound_moiety="LR Clonase(TM)"
                     /note="attR2"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    3389..3406
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     misc_feature    3407..3408
                     /note="multiple cloning region"
     regulatory      3471..3692
                     /note="SV40 late polyA signal"
     regulatory      3791..4203
                     /note="SV40 early enhancer/promoter"
     gene            4234..5271
                     /gene="hygR"
                     /note="hygR"
     CDS             4234..5271
                     /codon_start=1
                     /transl_table=11
                     /gene="hygR"
                     /product="hygromycin resistance protein"
                     /note="hygR"
                     /protein_id="AHH41357.1"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     regulatory      5295..5343
                     /note="synthetic polyA signal"
     gene            5604..6464
                     /gene="ampR"
                     /note="ampR"
     CDS             5604..6464
                     /codon_start=1
                     /transl_table=11
                     /gene="ampR"
                     /product="beta-lactamase"
                     /function="resistance to ampicillin"
                     /note="ampR"
                     /note="ampicillin-resistance protein; removed RE site from 
                     beta-lactamase gene"
                     /protein_id="AHH41359.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6619..6655
                     /note="ColE1-derived plasmid replication origin"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tggtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt attaatacga ctcactatag ggctagcgct caccatggtc ttcacactcg
     1081 aagatttcgt tggggactgg cgacagacag ccggctacaa cctggaccaa gtccttgaac
     1141 agggaggtgt gtccagtttg tttcagaatc tcggggtgtc cgtaactccg atccaaagga
     1201 ttgtcctgag cggtgaaaat gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag
     1261 gtctgagcgg cgaccaaatg ggccagatcg aaaaaatttt taaggtggtg taccctgtgg
     1321 atgatcatca ctttaaggtg atcctgcact atggcacact ggtaatcgac ggggttacgc
     1381 cgaacatgat cgactatttc ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa
     1441 agatcactgt aacagggacc ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca
     1501 accccgacgg ctccctgctg ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt
     1561 gcgaacgcat tctggcgggc tcgagcggcg cgatcgcttc cgaattcaga gctcaaccgc
     1621 ggatatctag atttgggccc aattcctgca ggattttgcg gccgcggatc tccacaagtt
     1681 tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga
     1741 ttttgcataa aaaacagact acataatact gtaaaacaca acatatccag tcactatggc
     1801 ggccgcatta ggcaccccag gctttacact ttatgcttcc ggctcgtata atgtgtggat
     1861 tttgagttag gatccgtcga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat
     1921 cactggatat accaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt
     1981 tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt
     2041 aaagaccgta aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg
     2101 cctgatgaat gctcatccgg aattccgtat ggcaatgaaa gacggtgagc tggtgatatg
     2161 ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct
     2221 ctggagtgaa taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc
     2281 gtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt
     2341 ctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa
     2401 cttcttcgcc cccgttttca ccatgggcaa atattatacg caaggcgaca aggtgctgat
     2461 gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct
     2521 taatgaatta caacagtact gcgatgagtg gcagggcggg gcgtaaacgc gtggatccgg
     2581 cttactaaaa gccagataac agtatgcgta tttgcgcgct gatttttgcg gtataagaat
     2641 atatactgat atgtataccc gaagtatgtc aaaaagaggt atgctatgaa gcagcgtatt
     2701 acagtgacag ttgacagcga cagctatcag ttgctcaagg catatatgat gtcaatatct
     2761 ccggtctggt aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc gaacgctgga
     2821 aagcggaaaa tcaggaaggg atggctgagg tcgcccggtt tattgaaatg aacggctctt
     2881 ttgctgacga gaacaggggc tggtgaaatg cagtttaagg tttacaccta taaaagagag
     2941 agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc cgggcgacgg
     3001 atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac
     3061 ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg
     3121 ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa
     3181 aacgccatta acctgatgtt ctggggaata taaatgtcag gctcccttat acacagccag
     3241 tctgcaggtc gaccatagtg actggatatg ttgtgtttta cagtattatg tagtctgttt
     3301 tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt ctcgttcagc
     3361 tttcttgtac aaagtggtaa gtaactaaga tccatcgagc tcgagcgctt gctgagttgg
     3421 ctgctgccac cgctgagcaa taactagcat aaccccttgg ccgcttcgag cagacatgat
     3481 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     3541 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     3601 taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt gggaggtttt
     3661 tttaagcaag taaaacctct acaaatgtgg taaaatcgaa ttttaacaaa atattaacgc
     3721 ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3781 acgcggatct gcgcagcacc atggcctgaa ataacctctg aaagaggaac ttggttaggt
     3841 accttctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     3901 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     3961 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4021 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4081 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4141 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4201 aaaaagctcg attcttctga cactagcgcc accatgaaga agcccgaact caccgctacc
     4261 agcgttgaaa aatttctcat cgagaagttc gacagtgtga gcgacctgat gcagttgtcg
     4321 gagggcgaag agagccgagc cttcagcttc gatgtcggcg gacgcggcta tgtactgcgg
     4381 gtgaatagct gcgctgatgg cttctacaaa gaccgctacg tgtaccgcca cttcgccagc
     4441 gctgcactac ccatccccga agtgttggac atcggcgagt tcagcgagag cctgacatac
     4501 tgcatcagta gacgcgccca aggcgttact ctccaagacc tccccgaaac agagctgcct
     4561 gctgtgttac agcctgtcgc cgaagctatg gatgctattg ccgccgccga cctcagtcaa
     4621 accagcggct tcggcccatt cgggccccaa ggcatcggcc agtacacaac ctggcgggat
     4681 ttcatttgcg ccattgctga tccccatgtc taccactggc agaccgtgat ggacgacacc
     4741 gtgtccgcca gcgtagctca agccctggac gaactgatgc tgtgggccga agactgtccc
     4801 gaggtgcgcc acctcgtcca tgccgacttc ggcagcaaca acgtcctgac cgacaacggc
     4861 cgcatcaccg ccgtaatcga ctggtccgaa gctatgttcg gggacagtca gtacgaggtg
     4921 gccaacatct tcttctggcg gccctggctg gcttgcatgg agcagcagac tcgctacttc
     4981 gagcgccggc atcccgagct ggccggcagc cctcgtctgc gagcctacat gctgcgcatc
     5041 ggcctggatc agctctacca gagcctcgtg gacggcaact tcgacgatgc tgcctgggct
     5101 caaggccgct gcgatgccat cgtccgcagc ggggccggca ccgtcggtcg cacacaaatc
     5161 gctcgccgga gcgcagccgt atggaccgac ggctgcgtcg aggtgctggc cgacagcggc
     5221 aaccgccggc ccagtacacg accgcgcgct aaggaggtag gtcgagttta gactctagcc
     5281 atcacgatgg ccgcaataaa atatctttat tttcattaca tctgtgtgtt ggttttttgt
     5341 gtgaatcgat agcgataagg atcctctttg cgcttgcgtt ttcccttgtc cagatagccc
     5401 agtagctgac attcatccgg ggtcagcacc gtttctgcgg actggctttc tacgtaatgg
     5461 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     5521 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     5581 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5641 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5701 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     5761 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact ttcaaagttc
     5821 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5881 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5941 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     6001 ccaacttact tctgacaact atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     6061 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6121 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     6181 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6241 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6301 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     6361 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6421 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaattcgaa atgaccgacc
     6481 aagcgacgcc caaccggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     6541 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     6601 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     6661 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     6721 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     6781 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     6841 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6901 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6961 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     7021 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     7081 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     7141 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     7201 aggatttcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     7261 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     7321 atagtccgga aatacaggaa cgcacgctgg atggcccttc gctgggatgg tgaaaccatg
     7381 aaaaatggca gcttcagtgg attaagtggg ggtaatgtgg cctgtaccct ctggttgcat
     7441 aggtattcat acggttaaaa tttatcaggc gcgattgcgg cagtttttcg ggtggtttgt
     7501 tgccattttt acctgtctgc tgccgtgatc gcgctgaacg cgttttagcg gtgcgtacaa
     7561 ttaagggatt atggtaaatc cacttactgt ctgccctcgt agccatcgag ataaaccgca
     7621 gtactccggc cacgatgcgt ccggcgtaga ggatcgagat ct
//
